Cloning and expression of biologically active α-galactosidase A

ABSTRACT

The present invention involves the production of large quantities of human α-Gal A by cloning and expressing the α-Gal A coding sequence in eukaryotic host cell expression systems. The eukaryotic expression systems, and in particular the mammalian host cell expression system described herein provide for the appropriate cotranslational and posttranslational modifications required for proper processing, e.g., glycosylation, phosphorylation, etc. and sorting of the expression product so that an active enzyme is produced. In addition, the expression of fusion proteins which simplify purification is described. 
     Using the methods described herein, the recombinant α-Gal A is secreted by the engineered host cells so that it is recovered from the culture medium in good yield. The α-Gal A produced in accordance with the invention may be used, but is not limited to, in the treatment in Fabry Disease; for the hydrolysis of α-galactosyl residues in glycoconjugates; and/or for the conversion of the blood group B antigen on erythrocytes to the blood group O antigen.

This is a Continuation-In-Part of Ser. No. 602,824 filed Oct. 24, 1990and Ser. No. 07/602,608 filed Oct. 24, 1990, each of which isincorporated by reference herein in its entirety.

TABLE OF CONTENTS

1. Introduction

2. Background Of The Invention

2.1. α-GAL A And Fabry Disease

2.2. The α-Gal A Enzyme

2.3. Lysosomal Enzymes:Biosynthesis And Targeting

3. Summary Of The Invention

3.1. Definitions

4. Description Of The Figures

5. Detailed Description Of The Invention

5.1. The α-GAL A Coding Sequence

5.2. Production Of Recombinant α-Gal A

5.2.1. Construction Of Expression Vectors And Preparation OfTransfectants

5.2.2. Identification Of Transfectants Or Transformants Expressing Theα-Gal A Product

5.2.3. Purification Of The α-GAL A Gene Product

5.2.4. Characterization Of The Recombinant Enzyme

5.2.5. Modified Glycoforms Of Recombinant α-Gal A For Enzyme Therapy InFabry Disease

5.3. Uses Of The Recombinant α-Gal A

5.3.1. α-Gal A Enzyme Therapy In Fabry Disease

5.3.2. In Vitro Uses of α-Gal A

6. Example:Overexpression And Specific Secretion Of Biologically Activeα-Galactosidase A In A Mammalian Cell System

6.1. Materials And Methods

6.1.1. Materials

6.1.2. Construction Of Expression Vector p91-AGA

6.1.3. Cell Culture, Electrotransfection, And Gene Amplication

6.1.4. Enzyme And Protein Assays

6.2. Results

6.2.1. Expression Of Human α-Gal A In COS-1 Cells

6.2.2. Transfection And Amplification Of α-Gal A In dhfr CHO Cells

6.2.3. High Level Expression Clones Secrete Human α-Gal A

6.2.4. Specific Secretion Of Over-Expressed Lysosomal Enzymes

6.2.5. Effect Of Serum Concentration On Secretion

6.2.6. Production In Bioreactors

6.3. Discussion

7. Example: Purification, Characterization And Processing Of Recombinantα-Galactosidase A

7.1. Materials And Methods

7.1.1. Materials

7.1.2. Cell Culture

7.1.3. Purification Of Recombinant α-Gal A

7.1.4. Enzyme And Protein Assays

7.1.5. In Vivo Natural Substrate Assay

7.1.6. Polyclonal Antibodies

7.1.7. SDS-Page And Autoradiography

7.1.8. Isoelectric Point And pH Optimum Determination

7.1.9. Mannose-6-Phosphate Receptor Affinity Chromatography And QAESephadex Chromatography

7.1.10. Labeling Of Cells With [³⁵ S]-Methionine, [³ H]-Mannose And [³²P]-Phosphorous

7.1.11. Cell Lysis And Immunoprecipitation

7.2. Results

7.2.1. Purification

7.2.2. Physicokinetic Properties

7.2.3. Processing And Rate Of Secretion Of Recombinant α-Gal A

7.2.4. Analysis Of Carbohydrate Moieties On Recombinant α-Gal A

7.2.5. Phosporylation

7.2.6. Analysis Of Endo H Sensitive Oligosaccharides

7.2.7. Interaction Of α-Gal A With The Mannose-6-Phosphate Receptor

7.2.8. Receptor Mediated Uptake Of Recombinant α-Gal A In FabryFibroblasts

8. Example: α-Gal A-Protein A Fusion Expressed In Mammalian Cells

8.1. Materials And Methods

8.1.1. Materials

8.1.2. Cell Culture And Transfections

8.1.3. PCR, DNA Sequencing And Vector Constructions

8.2. Results

8.2.1. Construction Of α-Gal A-Protein A (AGA-PA) Fusion

8.2.2. Expression Of pAGA-PA In COS-1 Cells

8.2.3. Affinity Purification Of α-Gal A

9. Example: In Vivo Modification Of Recombinant Human α-Gal AGlycosylation By α 2, 6-Sialyltransferase

9.1. Materials And Methods

9.1.1. Construction Of The α 2,6-Sialyltransferase Expression Vector,pST26

9.1.2. SNA-Lectin Fluorescence Microscopy

9.1.3. Purification Of Radiolabelled Human α-GAL A With And Withoutα2,6-Sialic Acid Residues

9.1.4. In Vivo Half-Life And Tissue Distribution Of Recombinant α-GAL A

9.2. Results

9.2.1. Introduction Of PST26 Into DG5.3-1000Mx CHO Cells AndDemonstration Of α2,6-Sialyltransferase Activity In G418-Selected Clones

9.2.2. Characterization Of Recombinant Human Secreted α2,6-Sialylatedα-GAL A

9.2.3. Plasma Half-Life And Tissue Distribution Of α2,6-Sialylated Humanα-GAL A In Mice

10. Example: Overexpression Of Human Lysosomal Proteins Results In TheirIntracellular Aggregation, Crystallization In Lysosomes, And SelectiveSecretion

10.1. Materials And Methods

10.1.1. Construction Of Plasmids For Lysosomal Enzyme Overproduction

10.1.2. Cell Culture, Electrotransfection, And Gene Amplification

10.1.3. Ultrastructural And Immunolabeling Studies

10.1.4. SDS-Page And Autoradiography

10.1.5. In Vitro Studies Of α-GAL A Aggregation

10.1.6. Enzyme And Protein Assays

10.2. Results

10.2.1. Overexpression Results In Crystalline Structures ContainingHuman α-GAL A In Membrane-Limited Vesicles

10.2.2. α-GAL A And α-N-Acetylgalactosaminidase Aggregate At HighConcentration And Low pH

10.3. Discussion

11. Deposit Of Microorganisms

1. INTRODUCTION

The present invention relates to the production of biologically activehuman α-Galactosidase (α-Gal A) involving cloning and expression of thegenetic coding sequence for α-Gal A in eukaryotic expression systemswhich provide for proper post-translational modifications and processingof the expression product.

The invention is demonstrated herein by working examples in which highlevels of α-Gal A were produced in mammalian expression systems. Theα-Gal enzyme produced in accordance with the invention may be used for avariety of purposes, including but not limited to enzyme replacementtherapy for Fabry Disease, industrial processes involving the hydrolysisof α-D-galactosyl residues of glycoconjugates, and for the conversion ofthe blood group B antigen on erythrocytes to the blood group O antigen.

2. BACKGROUND OF THE INVENTION

In the early 1970's, several investigators demonstrated the existence oftwo α-Galactosidase isozymes designated A and B, which hydrolyzed theα-galactosidic linkages in 4-MU-and/or ρ-NP-α-D-galactopyranosides(Kint, 1971, Arch. Int. Physiol. Biochem. 79:633-644; Beutler & Kuhl,1972, Amer. J. Hum. Genet. 24:237-249; Romeo, et al., 1972, FEBS Lett.27:161-166; Wood & Nadler, 1972, Am. J. Hum. Genet. 24:250-255; Ho, etal., 1972, Am. J. Hum. Genet. 24:256-266; Desnick, et al., 1973, J. Lab.Clin. Med. 81:157-171; and Desnick, et al., 1989, in The Metabolic Basisof Inherited Disease, Scriver, C. R., Beaudet, A. L. Sly, W. S. andValle, D., eds, pp. 1751-1796, McGraw Hill, New York). In tissues, about80%-90% of total α-Galactosidase (α-Gal) activity was due to athermolabile, myoinositol-inhibitable α-Gal A isozyme, while arelatively thermostable, α-Gal B, accounted for the remainder. The two"isozymes" were separable by electrophoresis, isoelectric focusing, andion exchange chromatography. After neuraminidase treatment, theelectrophoretic migrations and pI value of α-Gal A and B were similar(Kint, 1971; Arch. Int. Physiol. Biochem. 79:633-644), initiallysuggesting that the two enzymes were the differentially glycosylatedproducts of the same gene. The finding that the purified glycoproteinenzymes had similar physical properties including subunit molecularweight (˜46 kDa), homodimeric structures, and amino acid compositionsalso indicated their structural relatedness (Beutler & Kuhl, 1972, J.Biol. Chem. 247: 7195-7200; Callahan, et al., 1973, Biochem. Med. 7:424-431; Dean, et al., 1977, Biochem. Biophys. Res. Comm. 77:1411-1417;Schram, et al., 1977, Biochim. Biophys. Acta. 482:138-144; Kusiak, etal., 1978, J. Biol. Chem. 253:184-190; Dean, et al., 1979, J. Biol.Chem. 254:10001-10005; and Bishop, et al., 1980, in Enzyme Therapy inGenetic Disease:2, Desnick, R. J., ed., pp. 17-32, Alan R. Liss, Inc.,New York). However, the subsequent demonstration that polyclonalantibodies against α-Gal A or B did not cross-react with the otherenzyme (Beutler & Kuhl, 1972, J. Biol. Chem. 247:7195-7200; and Schram,et al., 1977, Biochim. Biophys. Acta. 482:138-144); that only α-Gal Aactivity was deficient in hemizygotes with Fabry disease (Kint, 1971;Arch. Int. Physiol. Biochem. 79: 633-644; Beutler & Kuhl, 1972, Amer. J.Hum. Genet. 24:237-249; Romeo, et al., 1972, FEBS Lett. 27:161-166; Wood& Nadler, 1972, Am. J. Hum. Genet. 24:250-255; Ho, et al., 1972, Am. J.Hum. Genet. 24:256-266; Desnick, et al., 1973, J. Lab. Clin. Med.81:157-171; Desnick, et al., 1989, in The Metabolic Basis of InheritedDisease, Scriver, C. R., Beaudet, A. L. Sly, W. S. and Valle, D., eds,pp. 1751-1796, McGraw Hill, New York; and, Beutler & Kuhl, 1972, J.Biol. Chem. 247:7195-7200); and that the genes for α-Gal A and B mappedto different chromosomes (Desnick, et al., 1989, in The Metabolic Basisof Inherited Disease, Scriver, C. R., Beaudet, A. L. Sly, W. S. andValle, D., eds, pp. 1751-1796, McGraw Hill, New York; deGroot, et al.,1978, Hum. Genet. 44:305-312), clearly demonstrated that these enzymeswere genetically distinct.

2.1. α-GAL A AND FABRY DISEASE

In Fabry disease, a lysosomal storage disease resulting from thedeficient activity of α-Gal A, identification of the enzymatic defect in1967 (Brady, et al., 1967, N. Eng. J. Med. 276:1163) led to the first invitro (Dawson, et al., 1973, Pediat. Res. 7: 694-690m) and in vivo(Mapes, et al., 1970, Science 169:987) therapeutic trials of α-Gal Areplacement in 1969 and 1970, respectively. These and subsequent trials(Mapes, et al., 1970, Science 169:987; Desnick, et al., 1979, Proc.Natl. Acad. Sci. USA 76: 5326; and, Brady, et al., 1973, N. Engl. J.Med. 289: 9) demonstrated the biochemical effectiveness of direct enzymereplacement for this disease. Repeated injections of purified splenicand plasma α-Gal A (100,000 U/injection) were administered to affectedhemizygotes over a four month period (Desnick, et al., 1979, Proc. Natl.Acad. Sci. USA 76:5326). The results of these studies demonstrated that(a) the plasma clearance of the splenic form was 7 times faster thanthat of the plasma form (10 min vs 70 min); (b) compared to the splenicform of the enzyme, the plasma form effected a 25-fold greater depletionof plasma substrate over a markedly longer period (48 hours vs 1 hour);(c) there was no evidence of an immunologic response to six doses ofeither form, administered intravenously over a four month period to twoaffected hemizygotes; and (d) suggestive evidence was obtainedindicating that stored tissue substrate was mobilized into thecirculation following depletion by the plasma form, but not by thesplenic form of the enzyme. Thus, the administered enzyme not onlydepleted the substrate from the circulation (a major site ofaccumulation), but also possibly mobilized the previously storedsubstrate from other depots into the circulation for subsequentclearance. These studies indicated the potential for eliminating, orsignificantly reducing, the pathological glycolipid storage by repeatedenzyme replacement.

However, the biochemical and clinical effectiveness of enzymereplacement in Fabry disease has not been demonstrated due to the lackof sufficient human enzyme for adequate doses and longterm evaluation.

2.2. THE α-Gal A ENZYME

The α-Gal A human enzyme has a molecular weight of approximately 101,000Da. On SDS gel electrophoresis it migrates as a single band ofapproximately 49,000 Da indicating the enzyme is a homodimer (Bishop &Desnick, 1981, J. Biol. Chem. 256: 1307). α-Gal A is synthesized as a50,500 Da precursor containing phosphorylated endoglycosidase Hsensitive oligosaccharides. This precursor is processed to a mature formof about 46,000 Da within 3-7 days after its synthesis. Theintermediates of this processing have not been defined (Lemansky, etal., 1987, J. Biol. Chem. 262:2062). As with many lysosomal enzymes,α-Gal A is targeted to the lysosome via the mannose-6-phosphatereceptor. This is evidenced by the high secretion rate of this enzyme inmucolipidosis II cells and in fibroblasts treated with NH₄ Cl.

The enzyme has been shown to contain 5-15% Asn linked carbohydrate(Ledonne, et al., 1983, Arch. Biochem. Biophys. 224:186). The tissueform of this enzyme was shown to have ˜52% high mannose and 48% complextype oligosaccharides. The high mannose type coeluted, on Bio-gelchromatography, with Man₈₋₉ GlcNAc while the complex typeoligosaccharides were of two categories containing 14 and 19-39 glucoseunits. Upon isoelectric focusing many forms of this enzyme are observeddepending on the sources of the purified enzyme (tissue vs plasma form).However, upon treatment with neuraminidase, a single band is observed(pI-5.1) indicating that this heterogeneity is due to different degreesof sialylation (Bishop & Desnick, 1981, J. Biol. Chem. 256:1307).Initial efforts to express the full-length cDNA encoding α-Gal Ainvolved using various prokaryotic expression vectors (Hantzopoulos andCalhoun, 1987, Gene 57:159; Ioannou, 1990, Ph.D. Thesis, City Universityof New York). Although microbial expression was achieved, as evidencedby enzyme assays of intact E. coli cells and growth on melibiose as thecarbon source, the human protein was expressed at low levels and couldnot be purified from the bacteria. These results indicate that therecombinant enzyme was unstable due to the lack of normal glycosylationand/or the presence of endogenous cytoplasmic or periplasmic proteases.

2.3. LYSOSOMAL ENZYMES: BIOSYNTHESIS AND TARGETING

Lysosomal enzymes are synthesized on membrane-bound polysomes in therough endoplasmic reticulum. Each protein is synthesized as a largerprecursor containing a hydrophobic amino terminal signal peptide. Thispeptide interacts with a signal recognition particle, an 11Sribonucleoprotein, and thereby initiates the vectoral transport of thenascent protein across the endoplasmic reticulum membrane into the lumen(Erickson, et al., 1981, J. Biol. Chem. 256:11224; Erickson, et al.,1983, Biochem. Biophys. Res. Commun. 115:275; Rosenfeld, et al., 1982,J. Cell Biol. 93:135). Lysosomal enzymes are cotranslationalyglycosylated by the en bloc transfer of a large preformedoligosaccharide, glucose-3, mannose-9, N-acetylglucosamine-2, from alipid-linked intermediate to the Asn residue of a consensus sequenceAsn-X-Ser/Thr in the nascent polypeptide (Kornfeld, R. & Kornfeld, S.,1985, Annu. Rev. Biochem. 54:631). In the endoplasmic reticulum, thesignal peptide is cleaved, and the processing of the Asn-linkedoligosaccharide begins by the excision of three glucose residues and onemannose from the oligosaccharide chain.

The proteins move via vesicular transport, to the Golgi stack where theyundergo a variety of posttranslational modifications, and are sorted forproper targeting to specific destinations: lysosomes, secretion, plasmamembrane. During movement through the Golgi, the oligosaccharide chainon secretory and membrane glycoproteins is processed to the sialicacid-containing complex-type. While some of the oligosaccharide chainson lysosomal enzymes undergo similar processing, most undergo adifferent series of modifications. The most important modification isthe acquisition of phosphomannosyl residues which serve as an essentialcomponent in the process of targeting these enzymes to the lysosome(Kaplan, et al., 1977, Proc. Natl. Acad. Sci. USA 74:2026). Thisrecognition marker is generated by the sequential action of two Golgienzymes. First, N-acetylglucosaminylphosphotransferase transfersN-acetylglucosamine-1-phosphate from the nucleotide sugar uridinediphosphate-N-acetylglucosamine to selected mannose residues onlysosomal enzymes to give rise to a phosphodiester intermediate (Reitman& Kornfeld, 1981, J. Biol. Chem. 256:4275; Waheed, et al., 1982, J.Biol. Chem. 257:12322). Then, N-acetylglucosamine-1-phosphodiesterα-N-acetylglucosaminidase removes N-acetylglucosamine residue to exposethe recognition signal, mannose-6-phosphate (Varki & Kornfeld, 1981, J.Biol. Chem. 256: 9937; Waheed, et al., 1981, J. Biol. Chem. 256:5717).

Following the generation of the phosphomannosyl residues, the lysosomalenzymes bind to mannose-6-phosphate (M-6-P) receptors in the Golgi. Inthis way the lysosomal enzymes remain intracellular and segregate fromthe proteins which are destined for secretion. The ligand-receptorcomplex then exits the Golgi via a coated vesicle and is delivered to aprelysosomal staging area where dissociation of the ligand occurs byacidification of the compartment (Gonzalez-Noriega, et al., 1980, J.Cell Biol. 85: 839). The receptor recycles back to the Golgi while thelysosomal enzymes are packaged into vesicles to form primary lysosomes.Approximately, 5-20% of the lysosomal enzymes do not traffic to thelysosomes and are secreted presumably, by default. A portion of thesesecreted enzymes may be recaptured by the M-6-P receptor found on thecell surface and be internalized and delivered to the lysosomes(Willingham, et al., 1981, Proc. Natl. Acad. Sci. USA 78:6967).

Two mannose-6-phosphate receptors have been identified. A 215 kDaglycoprotein has been purified from a variety of tissues (Sahagian, etal., 1981, Proc. Natl. Acad. Sci. USA, 78:4289; Steiner & Rome, 1982,Arch. Biochem. Biophys. 214:681). The binding of this receptor isdivalent cation independent. A second M-6-P receptor also has beenisolated which differs from the 215 kd receptor in that it has arequirement for divalent cations. Therefore, this receptor is called thecation-dependent (M-6-P^(CD)) while the 215 kd one is calledcation-independent (M-6-P^(CI)). The M-6-P^(CD) receptor appears to bean oligomer with three subunits with a subunit molecular weight of 46kDa.

3. SUMMARY OF THE INVENTION

The present invention involves the production of large quantities ofhuman α-Gal A by cloning and expressing the α-Gal A coding sequence ineukaryotic host cell expression systems. The eukaryotic expressionsystems, and in particular the mammalian host cell expression systemdescribed herein, provide for the appropriate cotranslational andposttranslational modifications required for proper processing, e.g.,glycosylation, sialylation, phosphorylation, etc. and sorting of theexpression product so that an active enzyme is produced. Also describedis the expression of α-galactosidase A fusion proteins which are readilypurified. These fusion proteins are engineered so that theα-galactosidase A moiety is readily cleaved from the fusion protein andrecovered.

Using the methods described herein, the recombinant α-Gal A is secretedby the engineered host cells so that it is recovered from the culturemedium in good yield. The α-Gal A produced in accordance with theinvention may be used for a variety of ends, including but not limitedto the treatment in Fabry Disease, the conversion of blood type B to O,or in any commercial process that involves the hydrolysis ofα-D-galactosyl residues from glycoconjugates.

Further, this invention describes a method whereby proteins that arenormally intracellularly targeted may be overexpressed and secreted fromrecombinant mammalian cell lines.

3.1. DEFINITIONS

As used herein, the following terms and abbreviations will have theindicated meaning:

    ______________________________________                                        Galactosidase A        α-Gal A                                          α-N-AcetylGalactosaminidase                                                                    α-GalNAc                                         base pair(s)           bp                                                     Chinese hamster ovary  CHO                                                    complementary DNA      cDNA                                                   counts per minute      cpm                                                    deoxyribonucleic acid  DNA                                                    Dulbecco's Modified Eagle's Medium                                                                   DMEM                                                   fetal calf serum       FCS                                                    kilobase pairs         kb                                                     kilodalton             kDa                                                    mannose-6-phosphate    M-6-P                                                  methotrexate           MTX                                                    4-methylumbelliferyl-α-D--galactoside                                                          4-MU-α-Gal                                       4-methyl-umbelliferyl-α-N-acetyl-                                                              4-Mu-α-GalNAc                                    galactosaminide                                                               micrograms             μg                                                  micrometer             μm                                                  nanograms              ng                                                     nanometer              nm                                                     nucleotide             nt                                                     p-nitrophenyl-α-N-Acetylgalactosaminide                                                        pNP-α-GalNAc                                     polyacrylamide gel electrophoresis                                                                   PAGE                                                   polymerase chain reaction                                                                            PCR                                                    ribonucleic acid       RNA                                                    sodium dodecyl sulfate SDS                                                    units                  U                                                      ______________________________________                                    

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Full-length human α-Gal cDNA sequence [SEQ ID NO:1].N-terminal, cyanogen bromide (CB), and tryptic (T) peptide amino acidsequences obtained from peptide microsequencing are indicated byunderlines [See SEQ ID NO:2]. Differences from the sequence predictedfrom the cDNA are shown. The four putative N-glycosylation sites aredenoted and the 3' termination signals are overlined.

FIGS. 1D-1F. Alignment of amino acid sequences deduced from thefull-length cDNAs encoding human α-Gal-NAc (α-Gal B), α-Gal-B) [SEQ. IDNO:3], α-Gal A [SEQ ID NO: 2], yeast Mel 1[SEQ ID NO:4], and E. coli MelA [SEQ ID NOS:5-7]. Colons, identical residues; single dots,isofunctional amino acids; and boxes, identical residues in α-GalNAc,α-Gal, Mel 1 and/or Mel A. Gaps were introduced for optimal alignment.Numbered vertical lines indicate exon boundaries for α-Gal (Bishop, etal, 1988, Proc. Natl. Acad. Sci. USA 85: 3903-3907).

FIG. 1G. Construction of the α-Gal mammalian expression vector p91-AGA.The full-length cDNA was excised from plasmid pcDAG126, adapted by theaddition of Eco RI linkers and subsequently cloned into the Eco RI siteof expression vector p91023(B).

FIG. 2. Transient expression of human α-Gal in COS-1 cells. Maximumactivity (U/mg) was reached 72 hours post-transfection in cellsreceiving the p91-AGA construct. No increase in α-Gal activity wasobserved in cells receiving no plasmid DNA nor in cells receiving thep91 vector with the α-Gal cDNA in the reverse orientation.

FIG. 3. Serum effect on secretion of recombinant α-Gal by CHO DG5.3.Cells were plated in DMEM supplemented with the appropriate serumconcentration (FIG. 3A. Cells were plated in DMEM supplemented with 10%FCS. Following confluency (.sup.˜ 4 days), the media was replaced withfresh DMEM supplemented with the appropriate serum concentration (FIG.3B).

FIG. 4. High-level production of recombinant α-Gal in a hollow fiberbioreactor. The amount of fetal bovine serum required by this system foroptimal cell-growth and protein secretion could be decreased to about1%.

FIG. 5. SDS-PAGE of each step of the α-Gal purification scheme. Lanes 1,6, molecular weight markers; lane 2, crude media; lane 3, affinitychromatography; lane 4, octyl-Sepharose chromatography; lane 5, superose6 chromatography.

FIG. 6. Total cellular (lanes 1-4) and media (lanes 5-8) from controlDG44 cells (lane 1,5), DG5 cells (lanes 2, 6), DG5.3 cells (lanes 3,7)and DG11 cells (lanes 4,8), labeled with [³⁵ S]-methionine.

FIGS. 7A-7C. Physicokinetic properties of recombinant α-Gal . Km towardsthe artificial substrate 4-MU-α-D-galactopyranoside (FIG. 7A).Isoelectric point of recombinant and human plasma purified enzyme (FIG.7B). pH optimum of the recombinant enzyme.

FIG. 8. P-C₁₂ STH degradation by CHO DG5.3 cells overproducing humanα-Gal . Rapid degradation of this substrate is observed by theaccumulation of P-C₁₂ SDH.

FIG. 9. Acquisition of disulfide bridges by recombinant α-Gal. CHO DG5.3cells were labeled with [³⁵ S]-methionine and chased for the indicatedtimes. SDS-PAGE in the absence of a reducing agent reveals the formationof secondary structures through disulfide bond formation.

FIG. 10. Arrival of newly synthesized α-Gal to the Golgi networkdetected by the acquisition of Endo H resistent oligosaccharides.

FIG. 11. Secretion rate of recombinant α-Gal. CHO DG5.3 cells werelabeled with [³⁵ S]-methionine for 5 min and chased with coldmethionine. Culture media aliquots were removed at the indicated timesand immunoprecipitated with anti-α-Gal polyclonal antibodies.

FIG. 12. SDS-PAGE of culture media from DG44 (lane 1; control), DG5(lane 2) and DG5.3 (lanes 3,4) cells labeled with [³⁵ S]-methionine for1 hour (lanes 1-3) and 24 hours (lane 4).

FIG. 13. Analysis of the carbohydrate moieties on recombinant α-Gal. CHODG5.3 cells were labeled with [³⁵ S]-methionine for 24 hours, theculture media collected and the recombinant enzyme immunoprecipitated.Aliquots were digested with endo D (lane 2), Endo H (lane 3), Endo F(lane 4), PNGase F (lane 5), Endo D and H (lane 6), Endo H and F (lane7), and Endo H, F, and PNGase F (lane 8). Untreated samples (lanes 1,9).

FIG. 14. Cellular (lanes 1,3) and secreted (lanes 2,4) forms ofrecombinant α-Gal treated with PNGase F (lanes 3,4). Controls (lanes1,2).

FIG. 15. Effect of glycosylation inhibitors on the secretion ofrecombinant α-Gal.

FIG. 16. ³² p labelling of CHO DG44 (lanes 2, 3) and DG5.3 (lanes 1, 4).α-Gal was immunoprecipitated from cells (lanes 1, 2) and media (lanes 2,3).

FIGS. 17A-17D. QAE-Sephadex chromatography of endo H sensitiveoligosaccharides of recombinant α-Gal. Untreated, dilute HCl treated,neuraminidase treated and alkaline phosphatase treated oligosaccharides.

FIG. 18. Endo H sensitive oligosaccharides of recombinant α-Galchromatographed on M-6-P receptor, solid circles, peak minus 4, opencircles, peak minus 2.

FIG. 19. Recombinant α-Gal chromatography on M-6-P receptor. DG5.3 cellslabeled with [³⁵ S]-methionine for 24 hours and media collected forchromatography. Solid circles, α-Gal activity; open boxes, totalradioactivity.

FIG. 20. Recombinant and human α-Gal affinity chromatography on M-6-Preceptor. Cells were labeled with [³⁵ S]-methionine for 24 hours in thepresence of NH₄ Cl and the culture media were collected. DG5.3secretions (FIG. 20A), MS914 secretions (FIG. 20B) and 293 secretions(FIG. 20C). Solid circles, α-Gal activity. Squares, total radioactivity.Open circles, M-6-P gradient used for elution.

FIG. 21. Uptake of recombinant α-Gal by Fabry fibroblasts. Cells wereincubated for the indicated amounts of α-Gal for 6 hours. Open circles,α-Gal uptake, closed circles, uptake in the presence of 2 mM M-6-P.

FIG. 22. Construction scheme of the α-Gal protein A fusion. The fusionwas accomplished in two separate PCR reactions as described in Materialsand Methods.

FIG. 23. Nucleotide sequence of the protein A domain E, collagenasecleavage sequence and 3'α-Gal sequence [SEQ ID NO:8]. Schematic of thefusion construct showing the collagenase consensus in relation to theα-Gal and protein A domains [SEQ ID NO:9].

FIG. 24. Plasma clearance following intravenous administration to miceof the recombinant human secreted α-galactosidase A containingα2,6-sialic acid residues (Δ--Δ) and the non-α2,6-sialylated glycoform(○--○) upper graph, compared with the plasma clearance of theseglycoforms following treatment with acid phosphatase. Each pointrepresents the average of the values from two independent injections.The T_(1/2) values were estimated by extrapolation.

FIG. 25. Tissue distribution of different forms of recombinant humansecreted α-Gal A following intravenous administration to mice. Eachpoint represents the average of the values from two independentinjections.

FIG. 26. DG5.3-1000Mx CHO Cells Contain Crystalline Structures ofOverexpressed Human α-Gal A. Electron micrographs of DG5.3-1000Mx cellsshowing crystalline structures in single membrane-limited vacuoles (A)and in vesicles, presumably in the dilated trans-Golgi (B; bar, 0.15 μmand 0.10 μm, respectively). (C and D) Immunoelectron microscopiclocalization of human α-Gal A with 10 nm colloidal gold particles (bar,0.31 μm snf 0.19 μm, respectively). (E) Electron micrograph of parentaldfhr DG44 cells (bar, 1.11 μm); inset showing Golgi complex (arrows) indfhr DG44 cells (bar, 0.5 μm).

FIG. 27. Aggregation of Purified Secreted α-Gal A is EnzymeConcentration and pH Dependent. (A) Precipitation of α-Gal A (10 mg/ml)with decreasing pH. Note that about 12% of incubated enzyme wasprecipitated at pH 6.0, the estimated pH of the trans-Golgi Network(TGN). (B) Turbidity of increasing concentrations of secreted α-Gal A atpH 5.0 (closed circles) and pH 7.0 (closed triangles) in the absence ofBovine Serum Albumin (BSA). As a control for non-specific precipitationwith increasing protein concentration, secreted α-Gal A (1 mg/ml) wasmixed with increasing BSA concentrations (0.1 to 10 mg/ml) at pH 5.0(solid squares). (C) SDS-PAGE of the supernatant and pellet fractionsfrom mixtures of purified secreted α-Gal A (10 mg/ml) and BSA (2 mg/ml)incubated at decreasing pH values. Note that α-Gal A was precipitatedwith decreasing pH whereas the concentrations of soluble andprecipitated BSA were essentially unchanged.

FIG. 28. Aggregation-Secretion Model for Selective Secretion of Humanα-Gal A Overexpressed in CHO Cells. High level overexpression in CHOcells of human α-Gal A or other lysosomal enzymes which normally aretargeted to the lysosome results in their selective secretion due totheir aggregation and the resultant inaccessibility of their M6PRsignals. The enzyme undergoes normal post-translational processing untilit arrives in the TGN, where the overexpressed enzyme undergoesprotein-protein interactions and forms smaller soluble and largerparticulate aggregates, due to lower pH of the TGN. The TGN becomesdilated with the overexpressed enzyme. Some aggregates and solubleenzyme with exposed M6P signals are trafficked to lysosomes, while themajority of aggregates whose M6P are not accessible are exocytosed bydefault via the constitutive secretory pathway. In addition, decreasedsulfation may occur as the tyrosines in the enzyme aggregates destinedfor secretion are unavailable to the sulfotransferase. This model mayexplain the selective secretion of other overexpressed proteins thatnormally are targeted to specific organelles.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the production of biologically activehuman α-Gal A involving cloning and expressing the nucleotide codingsequences for the enzyme in eukaryotic expression systems. Successfulexpression and production of this purified, biologically active enzymeas described and exemplified herein is particularly significant for anumber of reasons. For example, past efforts to express the full-lengthcDNA encoding α-Gal A using various prokaryotic expression vectorsresulted in expression of the enzyme, as evidenced by enzyme assays ofintact microbial host cells and growth on melibiose as the carbonsource; however, the human enzyme was expressed at low levels and couldnot be purified from the bacteria. These results indicate that therecombinant enzyme expressed in microbial systems was unstable due tothe lack of normal glycosylation and/or the presence of endogenouscytoplasmic or periplasmic proteases.

Efforts to express this enzyme in eukaryotic expression systems wereequally difficult for different reasons. The α-Gal A is a lysosomalenzyme encoded by a "housekeeping" gene. The primary translation productis highly modified and processed, requiring a complex series of eventsinvolving cleavage of a signal sequence, glycosylation, phosphorylation,and sialylation, which can be properly effected only by appropriate hostcells. Moreover, since the expression product is destined for thelysosome, which remains intracellular, it is quite surprising that themethods described herein allow for the secretion of a properlyprocessed, biologically active molecule.

The biologically active α-Gal A produced in accordance with theinvention has a variety of uses, probably the most significant being itsuse in enzyme replacement therapy for the lysosomal storage disorder,Fabry disease. For example, the metabolic defect in cultured fibroblastsfrom Fabry disease can be corrected in vitro by the addition ofexogenous α-Gal A into the culture medium. In addition, limited humantrials have demonstrated the biochemical effectiveness of enzymereplacement to deplete the circulating substrate prior to vasculardeposition. However, prior to the present invention, large quantities ofbiologically active, purified human α-Gal A could not be produced foruse in replacement therapies. The α-Gal A produced in accordance withthe invention also has a number of industrial uses, e.g., in any processinvolving the hydrolysis of α-D-galactosyl glycoconjugates, theconversion of blood group B to group O, etc., as described herein.

The invention is divided into the following sections solely for thepurpose of description: (a) the coding sequence for α-Gal A; (b)construction of an expression vector which will direct the expression ofthe enzyme coding sequence; (c) transfection of appropriate host cellswhich are capable of replicating, translating and properly processingthe primary transcripts in order to express a biologically active geneproduct; and (d) identification and/or purification of the enzyme soproduced. Once a transformant is identified that expresses high levelsof biologically active enzyme, the practice of the invention involvesthe expansion and use of that clone in the production and purificationof biologically active α-Gal A.

The invention is demonstrated herein, by way of examples in which cDNAsof α-Gal A were cloned and expressed in a mammalian expression system.Modifications to the cDNA coding sequences which improve yield, andsimplify purification without detracting from biological activity arealso described. Further, modifications to the host cells are describedthat allow for the expression of sialylated and asialylated glycoformsof the enzyme, both of which may be easily purified. Although theinvention is described for α-Gal A, the methods and modificationsexplained may be analogously applied to the expression of other secretedproteins, and in particular, other lysosomal enzymes, including but notlimited to α-N-acetylgalactosaminidase, and acid sphingomyelinase.

Various aspects of the invention are described in more detail in thesubsections below and in the examples that follow.

5.1. THE α-GAL A CODING SEQUENCE

The nucleotide coding sequence [SEQ ID NO:1] and deduced amino acidsequence [SEQ ID NO:2] for α-Gal A is depicted in FIG. 1A. Thisnucleotide sequence, or fragments or functional equivalents thereof, maybe used to generate recombinant DNA molecules that direct the expressionof the enzyme product, or functionally active peptides or functionalequivalents thereof, in appropriate host cells.

Due to the degeneracy of the nucleotide coding sequence, other DNAsequences which encode substantially the same amino acid sequences asdepected in FIG. 1A may be used in the practice of the invention for thecloning and expression of α-Gal A. Such alterations include deletions,additions or substitutions of different nucleotide residues resulting ina sequence that encodes the same or a functionally equivalent geneproduct. The gene product may contain deletions, additions orsubstitutions of amino acid residues within the sequence, which resultin a silent change thus producing a bioactive product. Such amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, the amphipathicnature of the residues involved and/or on the basis of crystallographicdata. For example, negatively charged amino acids include aspartic acidand glutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine;phenylalanine, tyrosine.

The coding sequences for α-Gal A may be conveniently obtained fromgenetically engineered microorganisms or cell lines containing theenzyme coding sequences, such as the deposited embodiments describedherein. Alternatively, genomic sequences or cDNA coding sequences forthese enzymes may be obtained from human genomic or cDNA libraries.Either genomic or cDNA libraries may be prepared from DNA fragmentsgenerated from human cell sources. The fragments which encode α-Gal Amay be identified by screening such libraries with a nucleotide probethat is substantially complementary to any portion of sequence ID NO:1depicted in FIG. 1A. Indeed, sequences generated by polymerase chainreaction can be ligated to form the full-length sequence. Althoughportions of the coding sequences may be utilized, full length clones,i.e., those containing the entire coding region for α-Gal A, may bepreferable for expression. Alternatively, the coding sequences depictedin FIG. 1A [SEQ ID NO:1] may be altered by the addition of sequencesthat can be used to increase levels of expression and/or to facilitatepurification. For example, as demonstrated in the working embodimentsdescribed herein, the α-Gal A coding sequence was modified by theaddition of the nucleotide sequence encoding the cleavage site forcollagenase followed by the Staphylococcal Protein A [SEQ ID NO:8].Exression of this chimeric gene construct resulted in a fusion proteinconsisting of α-Gal A--the collagenase substrate --Protein A [SEQ IDNO:9]. This fusion protein was readily purified using an IgG columnwhich binds to the Protein A moiety. Unfused α-Gal A was released fromthe column by treatment with collagenase which cleaved the α-Gal A fromthe Protein A moiety bound to the column. Other enzyme cleavagesubstrates and binding proteins can be engineered into similarconstructs for the production of α-Gal A which can be readily purifiedand released in its biologically active form.

Techniques well-known to those skilled in the art for the isolation ofDNA, generation of appropriate restriction fragments, construction ofclones and libraries, and screening recombinants may be used. For areview of such techniques, see, for example, Sambrook, et al., 1989,Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press,N.Y., Chapters 1-18.

In an alternate embodiment of the invention, the coding sequence of FIG.1A [SEQ ID NO:1] could be synthesized in whole or in part, usingchemical methods well-known in the art. See, for example, Caruthers, et.al., 1980, NUC. Acids Res. Symp. Ser. 7:215-233; Crea & Horn, 1980, Nuc.Acids Res. 9(10):2331; Matteucchi & Carruthers, 1980, TetrahedronLetters 21:719; and Chow and Kempe, 1981, Nuc. Acids Res.9(12):2807-2817.

Alternatively, the protein itself could be produced using chemicalmethods to synthesize the amino acid sequence depicted in FIG. 1A [SEQID NO:2] in whole or in part. For example, peptides can be synthesizedby solid phase techniques, cleaved from the resin and purified bypreparative high performance liquid chromatograph. (E.g., see,Creighton, 1983, Proteins, Structures and Molecular Principles, W. H.Freeman & Co., N.Y. pp. 50-60). The composition of the syntheticpeptides may be confirmed by amino acid analysis or sequencing (e.g.,the Edman degradation procedure; see Creighton, 1983, Proteins,Structures and Molecular Principles, W. H. Freeman & Co., N.Y., pp.34-49).

Human α-Gal A is a homodimeric glycoprotein. The full-length α-Gal AcDNA predicts a mature subunit of 398 amino acids. The amino acidsequence has an overall homology of about 50% with humanα-N-acetylgalactosaminidase (α-Gal B) [SEQ ID NO:3]. Homology searcheswith computerized data bases revealed short regions of α-Gal A homologywith the yeast Mel 1 [SEQ ID NO:4] and the E. coli Mel A [SEQ IDNOS:5-7] amino acid sequences (see FIG. 1B). It is likely that theseconserved regions are important for enzyme conformation, stability,subunit association and/or catalysis. Thus, it is preferred not to altersuch conserved regions. However, certain modifications in the codingsequence may be advantageous. For example, the four N-linkedglycosylation consensus sequences could be selectively obliterated,thereby altering the glycosylation of the enzyme and affectingphosphorylation, sialylation, sulfation, etc. Such modified enzymes mayhave altered clearance properties and targeting when injected into Fabrypatients.

Oligosaccharide modifications may be useful in the targeting of α-Gal Afor effective enzyme therapy. Some examples of such modifications aredescribed in more detail infra. Previous studies demonstrated that theplasma glycoform of α-Gal A, which is more highly sialylated than thesplenic glycoform, was more effective in depleting the toxic accumulatedcirculating substrate from Fabry patients (Desnick et al., 1977, Proc.Natl. Acad. Sci. USA 76:5326-5330). Studies characterizing the purifiedsplenic and plasma glycoforms of the enzyme revealed differences only intheir oligosaccharide moieties (Desnick et al., 1977, Proc. Natl. Acad.Sci. USA 76:5326-5330). Thus, efforts to target the recombinant enzymefor effective treatment of Fabry disease may be enhanced by modificationof the N-glycosylation sites.

Also, the 5' untranslated and coding regions of the nucleotide sequencecould be altered to improve the translational efficiency of the α-Gal AmRNA. For example, substitution of a cytosine for the guanosine inposition +4 of the α-Gal A cDNA could improve the translationalefficiency of the α-Gal A mRNA 5- to 10-fold (Kozak, 1987, J. Mol. Biol.196:947-950).

In addition, based on X-ray crystallographic data, sequence alterationscould be undertaken to improve protein stability, e.g., introducingdisulfide bridges at the appropriate positions, and/or deleting orreplacing amino acids that are predicted to cause protein instability.These are only examples of modifications that can be engineered into theα-Gal A enzyme to produce a more active or stable protein, more enzymeprotein, or even change the catalytic specificity of the enzyme.

5.2. PRODUCTION OF RECOMBINANT α-Gal A

In order to express a biologically active α-Gal A, the coding sequencefor the enzyme, a functional equivalent, or a modified sequence, asdescribed in Section 5.1., supra, is inserted into an appropriateeukaryotic expression vector, i.e., a vector which contains thenecessary elements for transcription and translation of the insertedcoding sequence in appropriate eukaryotic host cells which possess thecellular machinery and elements for the proper processing, i.e., signalcleavage, glycosylation, phosphorylation, sialylation, and proteinsorting. Mammalian host cell expression systems are preferred for theexpression of biologically active enzymes that are properly folded andprocessed; when administered in humans such expression products shouldexhibit proper tissue targeting and no adverse immunological reaction.

5.2.1. CONSTRUCTION OF EXPRESSION VECTORS AND PREPARATION OFTRANSFECTANTS

Methods which are well-known to those skilled in the art can be used toconstruct expression vectors containing the α-Gal A coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombination/genetic recombination. See, for example,the techniques described in Maniatis et al., 1982, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., Chapter 12.

A variety of eukaryotic host-expression systems may be utilized toexpress the α-Gal A coding sequence. Although prokaryotic systems offerthe distinct advantage of ease of manipulation and low cost of scale-up,their major drawback in the expression of α-Gal A is their lack ofproper post-translational modifications of expressed mammalian proteins.Eukaryotic systems, and preferably mammalian expression systems, allowfor proper modification to occur. Eukaryotic cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, phosphorylation, and, advantageously secretion of thegene product should be used as host cells for the expression of α-Gal A.Mammalian cell lines are preferred. Such host cell lines may include butare not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, WI38, etc.Alternatively, eukaryotic host cells which possess some but not all ofthe cellular machinery required for optional processing of the primarytranscript, and/or post-translational processing and/or secretion of thegene product may be modified to enhance the host cell's processingcapabilities. For example, a recombinant nucleotide sequence encoding apeptide product that performs a processing function the host cell hadnot previously been capable of may be engineered into the host cellline. Such a sequence may either be co-transfected into the host cellalong with the gene of interest, or included in the recombinantconstruct encoding the gene of interest. Alternatively, cell linescontaining this sequence may be produced which are then transfected withthe gene of interest.

Appropriate eukaryotic expression vectors should be utilized to directthe expression of α-Gal A in the host cell chosen. For example, at leasttwo basic approaches may be followed for the design of vectors on SV40.The first is to replace the SV40 early region with the gene of interestwhile the second is to replace the late region (Hammarskjold, et al.,1986, Gene 43:41). Early and late region replacement vectors can also becomplemented in vitro by the appropriate SV40 mutant lacking the earlyor late region. Such complementation will produce recombinants which arepackaged into infectious capsids and which contain the α-Gal A gene. Apermissive cell line can then be infected to produce the recombinantprotein. SV40-based vectors can also be used in transient expressionstudies, where best results are obtained when they are introduced intoCOS (CV-1, origin of SV40) cells, a derivative of CV-1 (green monkeykidney cells) which contain a single copy of an origin defective SV40genome integrated into the chromosome. These cells actively synthesizelarge T antigen (SV40), thus initiating replication from any plasmidcontaining an SV40 origin of replication.

In addition to SV40, almost every molecularly cloned virus or retrovirusmay be used as a cloning or expression vehicle. Viral vectors based on anumber of retroviruses (avian and murine), adenoviruses, vaccinia virus(Cochran, et al., 1985, Proc. Natl. Acad. Sci. USA 82:19) and polyomavirus may be used for expression. Other cloned viruses, such as J C(Howley, et al., 1980, J. Virol 36:878), BK and the human papillomaviruses (Heilman, et al., 1980, J. Virol 36:395), offer the potential ofbeing used as eukaryotic expression vectors. For example, when usingadenovirus expression vectors the α-Gal A coding sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing the human enzyme in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA)81:3655-3659). Alternatively, the vaccinia virus 7.5K promoter may beused. (e.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA)79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali etal., 1982, Proc. Natl. Acad. Sci. 79:4927-4931). Of particular interestare vectors based on bovine papilloma virus (Sarver, et al., 1981, Mol.Cell. Biol. 1:486). These vectors have the ability to replicate asextrachromosomal elements. Shortly after entry of this DNA into mousecells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA does not require integration of theplasmid into the host's chromosome, thereby yielding a high level ofexpression. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as the neo gene. High levelexpression may also be achieved using inducible promoters such as themetallothionine IIA promoter, heat shock promoters, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, following the introduction offoreign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with the α-Gal A or DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. A number of selectionsystems may be used, including but not limited to the herpes simplexvirus thymidine kinase (Wigler, et al., 1977, Cell 11:223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072; neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987,In: Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.).

Alternative eukaryotic expression systems which may be used to expressthe α-Gal A enzymes are yeast transformed with recombinant yeastexpression vectors containing the α-Gal A coding sequence; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the α-Gal A coding sequence; or plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the α-Gal A coding sequence.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol.II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of theYeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II. For complementation assays in yeast, cDNAs forα-Gal A may be cloned into yeast episomal plasmids (YEp) which replicateautonomously in yeast due to the presence of the yeast 2μ circle. ThecDNA may be cloned behind either a constitutive yeast promoter such asADH or LEU2 or an inducible promoter such as GAL (Cloning in Yeast,Chpt. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed.DM Glover, 1986, IRL Press, Wash., D.C.). Constructs may contain the 5'and 3' non-translated regions of the cognate α-Gal A mRNA or thosecorresponding to a yeast gene. YEp plasmids transform at high efficiencyand the plasmids are extremely stable. Alternatively, vectors may beused which promote integration of foreign DNA sequences into the yeastchromosome.

In cases where plant expression vectors are used, the expression of theα-Gal A coding sequence may be driven by any of a number of promoters.For example, viral promoters such as the 35S RNA and 19S RNA promotersof CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-Eor hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may beused. These constructs can be introduced into plant cells using Tiplasmids, Ri plasmids, plant virus vectors; direct DNA transformation;microinjection, electroporation, etc. For reviews of such techniquessee, for example, Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9.

An alternative expression system which could be used to express α-Gal Ais an insect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The α-Gal A sequence maybe cloned into non-essential regions (for example the polyhedrin gene)of the virus and placed under control of an AcNPV promoter (for examplethe polyhedrin promoter). Successful insertion of the coding sequencewill result in inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (E.g., see Smith et al., 1983, J. Viol. 46:584;Smith, U.S. Pat. No. 4,215,051).

5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSING THEα-Gal A PRODUCT

The host cells which contain the α-Gal A coding sequence and whichexpress the biologically active gene product may be identified by atleast four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b)the presence or absence of "marker" gene functions; (c) assessing thelevel of transcription as measured by the expression of α-Gal A mRNAtranscripts in the host cell; and (d) detection of the gene product asmeasured by immunoassay or by its biological activity.

In the first approach, the presence of the α-Gal A coding sequenceinserted in the expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the α-Gal A coding sequence [SEQ ID NO:1] substantially asshown in FIG. 1A, or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain "marker" gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the α-Gal A coding sequence is inserted within a marker gene sequenceof the vector, recombinants containing the α-Gal A coding sequence canbe identified by the absence of the marker gene function. Alternatively,a marker gene can be placed in tandem with the α-Gal A sequence underthe control of the same or different promoter used to control theexpression of the α-Gal A coding sequence. Expression of the marker inresponse to induction or selection indicates expression of the α-Gal Acoding sequence.

In the third approach, transcriptional activity for the α-Gal A codingregion can be assessed by hybridization assays. For example, RNA can beisolated and analyzed by Northern blot using a probe homologous to theα-Gal A coding sequence or particular portions thereof substantially asshown in FIG. 1A [SEQ ID NO:1]. Alternatively, total nucleic acids ofthe host cell may be extracted and assayed for hybridization to suchprobes.

In the fourth approach, the expression of the --Gal A protein productcan be assessed immunologically, for example by Western blots,immunoassays such as radioimmuno-precipitation, enzyme-linkedimmunoassays and the like. The ultimate test of the success of theexpression system, however, involves the detection of the biologicallyactive α-Gal A gene product. Where the host cell secretes the geneproduct, the cell free media obtained from the cultured transfectanthost cell may be assayed for α-Gal A activity. Where the gene product isnot secreted, cell lysates may be assayed for such activity. In eithercase, a number of assays can be used to detect α-Gal A activityincluding but not limited to: (a) assays employing the syntheticfluorogenic or chromogenic α-D-galactosides such as4-methylumbelliferyl-α-D-galactopyranoside (Desnick et al., 1973, J.Lab. Clin. Invest. 81:157); (b) assays employing the radiolabeled orfluorescent labeled natural substrates such as tritiated globotriaosylceramide or pyrene-dodecanoyl-sphingosine-trihexoside (Bishop andDesnick, 1981, J. Biol. Chem. 256:1307); and (c) assays employingX-α-gal.

5.2.3. PURIFICATION OF THE α-GAL A GENE PRODUCT

Once a clone that produces high levels of biologically active α-Gal A isidentified, the clone may be expanded and used to produce large amountsof the enzyme which may be purified using techniques well-known in theart including, but not limited to immunoaffinity purification,chromatographic methods including high performance liquid chromatographyand the like. Where the enzyme is secreted by the cultured cells, α-GalA may be readily recovered from the culture medium.

As demonstrated in the working examples described infra, recombinantα-Gal A was purified from the crude media by affinity chromatography onα-GalNH₂ -C₁₂ -Sepharose followed by hydrophobic chromatography on OctylSepharose and gel filtration on a 100 cm Superose 6 column. Therecombinant enzyme was essentially homogeneous following the gelfiltration step and was >98% pure as judged by SDS-PAGE.

Human recombinant α-Gal A was purified to homogeneity from the media ofthe CHO cell line, DG5.3, which was shown to secrete most of therecombinant enzyme. The culture media from this clone was highlyenriched for α-Gal A when serum-free medium was used, constitutinggreater than 95% of the total extracellular protein. Thus, purificationto homogeneity could be accomplished in only three chromatographicsteps. Over half a gram of enzyme was produced in three months and froma portion of this, 280 mg was purified with a yield of 80% using onlylaboratory-scale equipment. Notably, the recombinant enzyme had fullenzymatic activity with a specific activity equal to that of-thepreviously purified human enzyme (Bishop, et al., 1978, Biochim.Biophys. Acta. 525:399; Bishop and Desnick, 1981, J. Biol. Chem.256:1307). The recombinant enzyme was able to recognize and effectivelycleave an analog of the natural substrate, globotriaosylceramide.

Where the α-Gal A coding sequence is engineered to encode a cleavablefusion protein, the purification of α-Gal A may be readily accomplishedusing affinity purification techniques. In the working examplesdescribed infra, a collagenase cleavage recognition consensus sequencewas engineered between the carboxy terminus of α-Gal A and protein A.The resulting fusion protein [SEQ ID NO:9 was readily purified using anIgG column that bound the protein A moiety. Unfused α-Gal A was readilyreleased from the column by treatment with collagenase.

In particular, the overlap extension method (Ho, et al., 1989, Gene77:51; Kadowaki, et al., 1989, Gene 76:161) was used to fuse thefull-length α-Gal A cDNA to the protein A domain E of Staphylococcusaureus. Following transfection by electroporation, the α-Gal A activityin COS-1 cell extracts was increased 6 to 7-fold. In addition, thetransfected cells secreted significant amounts of the fusion proteininto the culture media (400 U/ml). The secreted fusion protein wasrapidly purified by a single IgG affinity purification step. Theengineering of a collagenase cleavage recognition consensus sequencebetween these two polypeptides facilitated the cleavage of the fusionprotein so that the purified human α-Gal A polypeptide could be readilyseparated from the protein A domain by a second IgG purification step.Of interest was the fact that the fusion construct retained α-Galactivity, presumably indicating that the enzyme polypeptide formed theactive homodimeric configuration even though the carboxy terminus wasjoined to an additional 56 residues of the protein A domain. Since COS-1cells transfected with an α-Gal A construct exhibit similar levels ofexpression and distribution between cells and media it appears that theprotein A domain does not interfere with either the folding or theproper processing of this lysosomal enzyme. Furthermore, the presence ofthe dimerized α-Gal A polypeptide did not inhibit the binding of theprotein A domain to the IgG affinity column. The insertion of the fourresidue collagenase cleavage recognition sequence between the α-Gal Aand protein A polypeptides permited cleavage of the fusion proteinleaving only two of the collagen residues on each of the peptides.

The ease of cDNA construction using the polymerase chain reaction,transfection and purification of the expressed protein permits theisolation of small, but sufficient amount of α-Gal A forcharacterization of the enzyme's physical and kinetic properties. Usingsite-directed mutagenesis or naturally occuring mutant sequences, thissystem provides a reasonable approach to determine the effects of thealtered primary structure on the function of the protein. Fusionconstructs with the protein A domain E preceeding the amino terminus andthe following the carboxy terminus may also be engineered to evaluatewhich fusion construct will interfere the least, if at all, with theprotein's biologic function and the ability to bind IgG.

Using this aspect of the invention, any cleavage site or enzyme cleavagesubstrate may be engineered between the α-Gal A sequence and a secondpeptide or protein that has a binding partner which could be used forpurification, e.g., any antigen for which an immunoaffinity column canbe prepared.

5.2.4. CHARACTERIZATION OF THE RECOMBINANT ENZYME

The purified recombinant enzyme produced in the mammalian expressionsystems described herein (e.g., the CHO expression system), hadmolecular weight, pH optimum, km and isoelectric point values which wereessentially identical to those of the enzyme purified from the humanplasma (Bishop, et al., 1978, Biochim. Biophys. Acta. 525:399; Bishopand Desnick, 1981, J. Biol. Chem. 256:1307). Analysis of thecarbohydrate moieties on this enzyme revealed the presence of threeoligosaccharide chains on the α-Gal A polypeptide. These chains were amixture of complex, hybrid and high-mannose types as evidenced byendoglycosidase and QAE Sephadex studies. Most importantly, therecombinant enzyme was also similar to the native plasma form of α-Gal Ain having terminal sialic acid moieties (Bishop & Desnick, 1981, J Biol.Chem. 256: 1307). In the limited clinical trial described supra, theplasma form of the enzyme was shown to be more effective in degradingcirculating GbOse₃ Cer than the splenic form. Therefore, the recombinantenzyme or a modified recombinant enzyme, including but not limited tomodifications of its carbohydrate chains or amino acid sequence, may bethe most appropriate form for enzyme replacement therapy of Fabrydisease. Indeed, the saturable uptake of recombinant α-Gal A by Fabryand normal fibroblasts is demonstrated in the examples herein, and isshown to be specifically inhibited by 2 mMmannose-6-phosphate.

In addition, the CHO expression system described herein has greatpromise for studies of the cell biology of lysosomal biogenesis andglycohydrolase processing. Light microscopy revealed highly vacuolatedcytoplasm in the DG5.3 CHO cells suggesting a proliferation of lysosomalmembranes and offering the potential for analysis of lysosomalbiogenesis. Preliminary studies have indicated that the recombinantenzyme is synthesized very rapidly, exits the endoplasmic reticulum in5-10 min following its synthesis and is secreted 45-60 min later. Thesefast kinetics of recombinant α-Gal A biosynthesis allow for interestingstudies involving lysosomal enzyme biosynthesis and offer a methodologythat, to date, is only rivaled by viral systems. In fact, recombinantα-Gal A is synthesized so rapidly that a single radioactive pulse of 3min is sufficient to label enough enzyme for these studies. Theunexpectedly specific secretion of only the overproduced recombinantα-Gal A and not other lysosomal enzymes appears analogous to "genedosage-dependant secretion" described by Rothman, et al. (Stevens etal., 1986, J. Cell Biol. 102:1551; Rothman et al., 1986, Proc. Natl.Acad. Sci. USA 83:3248 ) and poses interesting questions which can beevaluated in this system.

5.2.5. MODIFIED GLYCOFORMS OF RECOMBINANT α-Gal A FOR ENZYME THERAPY INFABRY DISEASE

Initial experiments to assess the clearance kinetics and tissuedistribution of recombinant α-Gal A in mice revealed 50% targeting tothe liver with the remaining enzyme being distributed to many othertissues including significant targeting to kidney, heart and skin. Whilethis distribution is similar to that previously observed for the plasmaform of human α-Gal A in mice, it may be appropriate to modify theenzyme for altered tissue targeting. Modifications of the recombinantα-Gal A to enhance tissue targeting including selective deglycosylationof the complex and high mannose carbohydrate moieties covalentlyattached to the recombinant enzyme. In particular, the inventionincludes modification of host cells that allow for expression ofsialylated and asialylated glycoforms of the enzyme, both of which maybe easily purified (see Section 9 infra). For example, when using CHOcells to express α-Gal A, the CHO cells may be co-transfected withsialyl-transferase gene construct that supplies the missing function tothe CHO cell in order to express the sialylated glycoform of α-Gal A.

Alternatively, sequential deglycosylation to various glycoforms for usein the treatment of Fabry disease. Such modifications have proven to beimportant in effectively targeting β-glucocerebrosidase to macrophagesin the treatment of Gaucher disease (Barton, N. W., et al., 1990, Proc.Natl. Acad. Sci. USA 87:1913). In this case, placenta derivedβ-glucocerebrosidase was sequentially treated with neuraminidase,β-galactosidase and N-β-acetylglucosaminidase to expose terminal mannoseresidues for uptake by the mannose receptor of these cells (Stahl, etal., in The Molecular Basis of Lysosomal Disorders, Barranger, J. A. andBrady, R. O. eds., 1984 Academic Press, NY pp. 209-218).

Modifications to human recombinant α-Gal A included in the scope of thisinvention include, but are not limited to, sequential deglycosylation byneuraminidase to expose terminal galactose; β-galactosidase treatment toexpose N-β-acetylglucosaminyl residues; and N-β-acetylglucosaminidasetreatment to expose mannose residues for specific targeting and uptakeby various cell types. The sequentially deglycosylated recombinant α-GalA glycoforms may be analyzed by determining the clearance kinetics andtissue distribution of each of the radiolabeled glycoforms followingintravenous administration in mice and monkeys.

Deglycosylation of recombinant α-Gal A may be accomplished in a numberof ways. The general methods of sequential treatment by exo-glycosidaseswhich may be used are essentially those previously described (Murray, G.J., 1987, Meth. Enzymol, 149:25). For example, terminal sialic acidresidues can be removed by treatment with neuraminidase covalently boundto agarose; e.g., type VI neuraminidase attached to agarose (SIGMAChemical Co., St. Louis, Mo.) may be used at 40 U/g to treat 100 mgα-Gal A with 8 units of conjugated neuraminidase at pH 5.0 for 4 hour at37° C. The conjugated neuraminidase can be removed by centrifugation.Similarly, β-galactosidase (3 Units per 100 mg α-Gal A) purified fromStreptococcus pneumoniae may be used to remove terminal galactoseresidues. Finally, jack bean N-β-acetylglucosaminidase (SIGMA ChemicalCo., St. Louis, Mo.) can be used; e.g., 3×10⁶ units can be mixed witheach 100 mg aliquot of the recombinant α-Gal A for four hours at 37° C.At each step, the recombinant enzyme can be rapidly purified free ofdeglycosylating enzymes and free carbohydrate by purification over theα-galactosylamine-Sepharose affinity column.

For the analysis of the in vivo fate of the various glycoforms,including plasma clearance kinetics and tissue distribution studies, therecombinant α-Gal A may be labeled prior to modification. For example,the recombinant α-Gal A can be radiolabelled by growth in the CHO DG5.3cell line in the presence of 50 μCi/ml [³⁵ S]methionine (>1000 Ci/mmole)for 24 hours. The secreted radiolabeled enzyme can be purified from theharvested media by α-galactosylamine-Sepharose affinity chromatographyas previously described. Essentially 100% of the radiolabelled proteinsecreted by these cells is α-Gal A which can then be used for thesequential generation of the glycoforms.

5.3. USES OF THE RECOMBINANT α-Gal A

The purified products obtained in accordance with the invention may beadvantageously utilized for enzyme replacement therapy in patients withthe lysosomal storage disorder, Fabry Disease. Alternatively, thepurified products obtained in accordance with the invention may be usedin vitro to modify α-D-galacto-glyconjugates in a variety of processes;e.g,, to convert blood group B erythrocytes to blood group O; incommerical processes requiring the conversion of sugars such asraffinose to sucrose or melibiose to galactose and glucose; etc. Theseare discussed in more detail in the subsections below.

5.3.1. α-Gal A ENZYME THERAPY IN FABRY DISEASE

Among the inborn errors of metabolism, studies of patients withlysosomal storage disorders have provided basic understanding of thebiology of the lysosomal apparatus and its hydrolases, theirbiosynthesis and processing (Rosenfeld, et al., 1982, J. Cell Biol.93:135; Lemansky, et al., 1984, J. Biol. Chem. 259:10129), themechanisms of their transport to the lysosomes (Neufeld, et al., 1975,Ann. Rev. Biochem. 44:357; Sly et al., 1982, J. Cell Biochem. 18:67;Kornfeld, S., 1986, J. Clin. Invest. 77:1), and their cofactorrequirements (Verheijen, et al., 1985. Eur. J. Biochem. 149:315; d'Azzo,et al., 1982, Eur. J. Biochem. 149:315; Mehl, et al., 1964, Physiol.Chem. 339:260; Conzelman, et al., 1978, Proc. Natl. Acad. Sci. USA75:3979). Of the over 30 lysosomal storage disorders, Fabry disease isan ideal candidate for the application of the recombinant DNA techniquesdescribed herein to evaluate and utilize various therapeutic approachesin model systems, as well as to correlate the effects of site-specificchanges on enzyme structure and function. The disease has no centralnervous system involvement; thus, the blood/brain barrier does notpresent an obstacle to enzyme replacement therapy. The defective enzyme,α-Gal A, is a homodimer (Bishop & Desnick, 1981, J. Biol. Chem.256:1307), in contrast to some lysosomal enzymes which have differentsubunits such as β-hexosaminidase A (Mahuran, et al., 1982, Proc. Natl.Acad. Sci. USA 79:1602); therefore, only a single gene product must beobtained. The metabolic defect in cultured fibroblasts from Fabrydisease has been corrected in vitro by the addition of exogenous enzymeinto the culture medium (Cline, et al., 1986, DNA 5: 37). Also, atypicalvariants with Fabry disease have been identified, these males areclinically asymptomatic, having sufficient residual α-Gal A activity (3to 10%) to protect them from the major morbid manifestations of thedisease (Lemansky, et al., 1987, J. Biol. Chem. 262:2062; Clarke, etal., 1971, N. Engl. J. Med. 284:233; Romeo, et al., 1975, Biochem.Genet. 13:615; Bishop, et al., 1981, Am. J. Hum. Genet. 71:217A; Bach,et al., 1982, Clin. Genet. 21:59; and, Kobayashi, et al., 1985, J.Neurol. Sci. 67:179). Finally, as noted above, limited human trials havedemonstrated the biochemical effectiveness of enzyme replacement todeplete the circulating substrate prior to vascular deposition as wellas the absence of immunologic complications (Brady, et al., 1973, N.Engl. J. Med. 289:9; Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA76:5326; Bishop, et al., 1981, Enzyme Therapy XX: In: Lysosomes andLysosomal Storage Diseases, Callahan, J. W. and Lowden, J. A., (eds.),Raven Press, New York, pp. 381; Desnick, et al., 1980, Enzyme TherapyXVII: In: Enzyme Therapy in Genetic Disease: 2, Desnick, R. J. (ed.),Alan, R. Liss, Inc., New York, pp. 393).

In these studies, both splenic and plasma isoforms of the α-Gal A enzymewere administered intravenously. The circulating half-life of thesplenic isozyme was about 10 min whereas that for the plasma isozyme wasapproximately 70 min. After each dose of the splenic isozyme, theconcentration of the accumulated circulating substrate decreasedmaximally in 15 min. In contrast, injection of the plasma isozymedecreased circulating substrate levels gradually over 36-72 hours. Sincethe secreted form of the recombinant α-Gal A appears to be similar tothe plasma isozyme, the secreted form of the recombinant enzyme could beeffective for the long term depletion and control of circulatingsubstrate levels.

The dose of the partially purified plasma and splenic isozymesadministered in the above clinical trials was 2000 U/kg body weight, ora dose equivalent to giving 1 μg/kg of pure enzyme. Since this doseproved effective in reducing the level of circulating substrate, asimilar dose of the recombinant enzyme should have a similar effect.However, the recombinant enzyme could be administered at a dosageranging from 0.1 μg/kg to about 10 mg/kg and, preferably from about 0.1mg/kg to about 2 mg/kg. The ability to produce large amounts of therecombinant enzyme in accordance with this invention will permit theevaluation of the therapeutic effect of significantly larger doses.

5.3.2. IN VITRO USES OF α-Gal A

α-Gal A is a galactosyl hydrolase which has activity toward variousoligosaccharides, glycoproteins, glycopeptides and glycolipids withterminal α-galactosidic linkages. Thus, the enzyme can be used in vitroto modify these α-galactoglycoconjugates. For example, the recombinantα-Gal A of the invention could be utilized for a variety of desirablemodifications including but not limited to: (a) the conversion of bloodgroup B erythrocytes to cells expressing the blood group O antigen(Harpaz, et al., 1977, Eur. J. Biochem. 77:419-426); and (b) thehydrolysis of stacchyose to raffinose, raffinose to the disaccharidesucrose, or the hydrolysis of melibiose to galactose and glucose(Silman, et al., 1980, Biotechnol. Bioeng. 22:533). Such hydrolyses havecommercial applications as in the degradation of molasses as a substratefor yeast production (Liljestrom-Suominen, et al., 1988, Appl. Environ.Micro. 54:245-249).

6. EXAMPLE: OVEREXPRESSION AND SPECIFIC SECRETION OF BIOLOGICALLY ACTIVEα-GALACTOSIDASE A IN A MAMMALIAN CELL SYSTEM

The subsections below describe the production of large quantities ofhuman recombinant α-Gal A. A full-length cDNA encoding human α-Gal A wasinserted into the expression vector p91023(B) in front of theamplifiable dihydrofolate reductase (DHFR) cDNA. The functionalintegrity of cDNA construct (p91-AGA) was confirmed by transientexpression of active enzyme in COS-1 cells; 650 U/mg (nmol/hour) versusendogenous levels of ˜150 U/mg of 4-MU-α-D-galactopyranoside activity.The p91-AGA construct was introduced by electroporation into DG44 dhfr⁻CHO cells. Positive selection in media lacking nucleosides resulted inthe isolation of clones expressing the active enzyme at levels rangingfrom 300 to 2,000 U/mg. Selected subclones, grown in increasingconcentrations of methotrexate (MTX, 0.02 to 1.3 μM) to co-amplify DHFRand α-Gal A cDNAs, expressed intracellular levels of α-Gal A activityranging from 5,000 to 25,000 U/mg. Notably, subclone DG44.5, whichexpressed high intracellular levels of α-Gal A, secreted more than 80%of the total recombinant enzyme produced. At a MTX concentration of 500μM, 10⁷ cells secreted ˜15,000 U/ml culture media/day. Of note,endogenous CHO lysosomal enzymes were not secreted includingβ-hexosaminidase, α-mannosidase, β-galactosidase and β-glucuronidase,indicating that the secretion was α-Gal A specific and not due tosaturation of the mannose-6-phosphate receptor-mediated pathway. Using ahollow fiber bioreactor, up to 5 mg per liter per day of recombinantα-Gal A enzyme was produced per day. The secreted α-Gal A was purifiedby affinity chromatography for characterization of various physical andkinetic properties. The recombinant α-Gal A had a pI, electrophoreticmobility and Km values which were similar to the enzyme purified fromhuman plasma. In addition, ³² p labeling studies revealed that both thelysosomal and secreted forms were phosphorylated, presumably in theiroligosaccharide moieties. Current studies are directed to characterizeadditional kinetic and physical properties, the oligosaccharide moietiesand the crystal structure of the recombinant enzyme. Furthermore, theavailability of large amounts of soluble active enzyme will permit theevaluation of enzyme replacement in animal systems prior to clinicaltrials in hemizygotes with Fabry disease.

6.1. MATERIALS AND METHODS 6.1.1. MATERIALS

Restriction endonucleases, the Klenow fragment of DNA polymerase I, T4polymerase and T4 ligase were from New England Biolabs; α and γ-32[P]dNTPs (3000 Ci/mole) and α35[S]dATP (100 Ci/mole) were from Amersham.The COS-1 cell line was purchased from ATCC, Rockville, Md. The CHO DG44dhfr⁻ cell line is described (Urlaug, et al., 1986, Somat. Cell Genet.12:555-566).

6.1.2. CONSTRUCTION OF EXPRESSION VECTOR p91-AGA

Plasmid pcDAG126 (Bishop, et al., 1988, in, Lipid Storage Disorders,Salvaryre, R., Douste-Blazy, L. Gatt, S. Eds. Plenum PublishingCorporation, New York, pp. 809 to 822) containing the full-length α-GalA cDNA was digested with Bam HI and Pst I and the 1.45 kb insertfragment was purified by agarose gel elctrophoresis. The cDNA was thenforce-subcloned into plasmid pGEM-4 at the Bam HI and Pst I sitesresulting in pGEM-AGA126. This plasmid was then digested with Hind III,end-filled using Klenow and ligated to Eco RI linkers. After digestionwith Eco RI, the 1.45 kb fragment was purified as above and cloned intothe Eco RI site of the mammalian expression vector p91023(B) (Wong etal., 1985, Science 228:810) resulting in p91-AGA (FIG. 1C).

6.1.3. CELL CULTURE, ELECTROTRANSFECTION, AND GENE AMPLICATION

COS-1 and DG44 CHO cells were maintained at 37° C. in 5% CO₂ inDulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum(FCS) and antibiotics; DG44 (dhfr⁻) cells were maintained by addition of0.05 mM hypoxanthine and 0,008 mM thymidine to the media. Followingtransfection, the recombinant CHO lines were grown in DMEM supplementedwith 10% dialyzed FCS in the absence or presence of MTX.

For electroporation, cells were trypsinized and centrifuged at 800×g atroom temperature for 10 min. The pellet was washed once with DMEMsupplemented with 10% FCS serum and twice in ice-cold electroporationbuffer (phosphate buffered sucrose; 272 mM sucrose, 7 mM sodiumphosphate, pH 7.4, containing 1 mM MgCl₂). Cells were then resuspendedin phosphate buffered sucrose at ˜0.65 to 1.0×10⁷ /ml. The cellsuspension (0.8 ml) was placed in a 0.4 cm gap cuvette (Bio-Rad), 5-20μg of plasmid DNA was added and kept on ice for 10 min. The cuvette wasplaced in the "Gene Pulser" chamber (Bio-Rad) and pulsed once at 25 μFwith 300 V for COS-1 cells or 400 V for CHO DG44 (dhfr⁻) cells, theoptimized settings for the respective cell lines. The cuvette containingthe pulsed cells was placed on ice for 10 min and then the cells wereremoved from the cuvette and placed in 15 ml of DMEM supplemented with10% FCS.

For transient expression, COS-1 cells were harvested at 72 hours andassayed immediately. For stable expression, the transfected DG44 cellswere grown for 48 hours and then were removed from the culture dish bytrypsinization and replated at a 1:15 ratio in DMEM supplemented with10% dialyzed FCS. Media was replaced every four days. After two weeks ofgrowth, cell foci became visible and individual clones were isolatedwith cloning rings. Clones which expressed the highest levels of α-Gal Awere subjected to amplification en masse by step-wise growth inincreasing concentrations of methotrexate (MTX), 0.02, 0.08, 1.3, 20,40, 80, 250 and 500 μM.

6.1.4. ENZYME AND PROTEIN ASSAYS

For enzyme assay, the cells in a 100 mm culture dish were washed twicewith 5 ml of phosphate buffer saline (PBS) and scraped into a 12 mlconical tube using a rubber policeman. Following centrifugation at 800×gfor 10 min, the cells were resuspended in 1 ml of 25 mM NaPO₄ buffer, pH6.0, and then disrupted in a Branson cup sonicator with three 15 secondbursts at 70% output power. The sonicate was centrifuged at 10,000×g for15 min at 4° C. and the supernatant was removed and assayed immediately.Alternatively, for rapid screening, cells were washed as above and 1 mlof lysis buffer (50 mM sodium phosphate buffer, pH 6.5, containing 150mM NaCl, 1 mM EDTA, 1% NP-40, and 0.2 mM PMSF) was added to the dish.The lysed cells were incubated at 4° C. for 30 min, the lysatescollected and transferred to a 1.5 ml tube, centrifuged in a microfuge,and then the supernatant was removed for assay.

The α-Gal A activities in the cell lysates and media were determinedusing 5 mM 4-methylumbelliferyl-α-D-galactopyranoside (4MU-α-Gal) aspreviously described (Bishop, et al., 1980, In Enzyme Therapy in GeneticDiseases: 2. Desnick, R. J. (Ed.). Alan R. Liss, Inc. New York, p. 17).Briefly, a stock solution of 5 mM 4MU-α-Gal was prepared in 0.1Mcitrate/0.2M phosphate buffer, pH 4.6, in an ultrasonic bath. Thereaction mixture, containing 10 to 50 μl of cell extract and 150 μl ofthe stock substrate solution, was incubated at 37° C. for 10 to 30 min.The reaction was terminated with the addition of 2.3 ml of 0.1Methylenediamine. The fluorescence was determined using a Turner model111 Fluorometer. One unit of activity is the amount of enzyme whichhydrolyzes one nmol of substrate per hour. The activities ofα-mannosidase, β-galactosidase, β-hexosaminidase, β-glucuronidase andacid phosphatase were determined using the appropriate4-methylumbelliferyl substrate. Protein concentrations were determinedby the fluorescamine method (Bohlen, et al., 1973, Arch. Biochem.Biophys. 155:213) as modified by Bishop et al. (Bishop, et al., 1978,Biochim. Biophys. Acta 524:109).

6.2. RESULTS 6.2.1. EXPRESSION OF HUMAN α-Gal A IN COS-1 CELLS

The full-length human α-Gal A cDNA was cloned into the expression vectorp91023(B) (Wong, et al., 1985, Science 228:810) and the construct,designated p91-AGA, was introduced into COS-1 cells by electroporation.Increased levels of α-Gal A activity were detected at 24, 48 and 72hours after transfection (FIG. 2), indicating the functional integrityof the p91-AGA construct. At 72 hours after transfection, the α-Gal Aactivity increased about four-fold, while no increase in α-Gal Aactivity was observed in cells transfected with the p91023(B) vectorcontaining the α-Gal A cDNA in the antisense orientation, nor in thecells that received no DNA. In addition, the β-galactosidase levels,determined as a lysosomal enzyme control, were not changed.

6.2.2. TRANSFECTION AND AMPLIFICATION OF α-Gal A IN DHFR⁻ CHO CELLS

Recombinant clones stably expressing human α-Gal A were obtained byelectrotransfection of the p91-AGA construct into DG44 dhfr⁻ CHO cellsand amplification of the integrated vector DNA with selection inincreasing MTX concentrations. Initial growth in media lackingnucleosides resulted in the identification of over 100 clones expressingα-Gal A at levels ranging from 100 to 1,800 U/mg protein (Table I).Clones with the highest α-Gal A level were grown in the presence of 0.02to 0.08 μM MTX to amplify the integrated p91-AGA DNA. Table II showsthat the intracellular α-Gal A levels in representative amplified clonesincreased 2 to 6 -fol in 0.02 μM MTX and up to 10 fold when furtheramplified in 0.08 μM MTX.

                  TABLE I                                                         ______________________________________                                        Intracellular α-Galactosidase A Activity In                             DG 44 (dhfr.sup.-) CHO Cells* Following                                       Elecrotransfection with p91-AGA                                                              α-Gal A Activity                                         CLONE          (U/mg protein)                                                 ______________________________________                                        Parental DG44:   497                                                          Transfected:                                                                   4               493                                                           5             1,243                                                           7               108                                                           8               524                                                           9             1,155                                                          11             1,115                                                          20               624                                                          24             1,864                                                          46               720                                                          52               180                                                          ______________________________________                                         *Cells grown in DMEM supplemented with 10% dialyzed FCS.                 

                  TABLE II                                                        ______________________________________                                        Intracellular α-Galactosidase A Activities                              In p9l-AGA Transfected DG44 (dhfr.sup.-) CHO Cells                            Following Initial Applification In Methotrexate                                               α-Gal A                                                 CLONE           (U/mg)                                                        ______________________________________                                        0.02 μM MTX:                                                               5               4,990                                                         9               2,900                                                         11              3,170                                                         46.1            1,230                                                         46.5            4,570                                                         46.12           4,100                                                         0.08 μM MTX:                                                               5.3             23,400                                                        5.7             7,950                                                         5.9             14,680                                                        5.11            3,070                                                         9.1             10.290                                                        9.4             7,950                                                         9.6             3,860                                                         ______________________________________                                    

6.2.3. HIGH LEVEL EXPRESSION CLONES SECRETE HUMAN α-Gal A

Among the positive clones amplified in the presence of 0.08 μM MTX,clone 5.3 had the highest intracellular α-Gal A level (Table II) andtherefore was chosen for further amplification. When grown in thepresence of 1.3 μM MTX, the α-Gal A activity in the growth media ofclone DG5.3 was determined to be 2,500 U/ml, or 25-fold greater than thelevel in untransfected parental DG44 cells (50 to 100 U/ml). Growth inthe presence of increasing concentrations of MTX, resulted in increasedintracellular and secreted α-Gal A activities (Table III).Interestingly, over 80% of the total α-Gal A produced was secreted andgrowth in increasing MTX concentrations continued to increase thepercentage of enzyme secreted. Note that the data shown in Table IIIwere obtained after the cells were amplified in the presence of theindicated MTX concentration and then assayed for α-Gal A activity aftergrowth for three weeks in the absence of MTX, which accounts for theirlower intracellular activities than during growth under selectivepressure (Pallavicini, et al., 1990, Mol. Cell Biol. 10:401; Kaufman, R.J., 1990, Meth. Enzymol, 185:537; Kaufman, R. J., 1990, Meth. Enzymol,185:487).

                  TABLE III                                                       ______________________________________                                        Intracellular And Secreted α-Galactosidase A                            Activities In p91-AGA Transfected CHO Line DG5.3                              Following Step-Wise Amplification In Methotrexate.                            Data Were Obtained On Clones After Three Weeks Of                             Growth In The Absence Of Methotrexate.                                        Methotrexate                                                                  Concentration      CHO Cells Media*                                           (μm)            (U/mg)    (U/ml)                                           ______________________________________                                        Untransfected DG44:                                                                                250       100                                            Transfected p91AGA5-3:                                                        0.00                 375       150                                            0.02                 550       265                                            0.08                 600       560                                            1.3                2,560     2,090                                            20                 6,270     6,530                                            40                 5,795     6,855                                            80                 6,365     8,750                                            250                5,720     9,945                                            500                12,560    18,140                                           ______________________________________                                         *10.sup.7 cells and 10 ml of media for each Methotrexate concentration.  

6.2.4. SPECIFIC SECRETION OF OVER-EXPRESSED LYSOSOMAL ENZYMES

To determine whether the secretion of α-Gal A was due to saturation ofthe receptors for lysosomal targeting, the culture media from cloneDG5.3 was assayed for the presence of other lysosomal enzymes. As shownin Table IV, the activities of seven representative lysosomal enzymeswere not increased or were lower than those in the media of the DG44parental cell line, indicating that the DG5.3 secretion of α-Gal A wasspecific.

To determine if the secretion was specific to clone DG5.3, anotherclone, DG9, which was not secreting α-Gal A (i.e., activity in media was120 U/ml), was subjected to step-wise growth in increasing MTXconcentrations (i.e., from 0.02 to 20 μM MTX). After amplification in 20μM MTX, clone DG9 had intracellular and secreted levels of α-Gal Aactivity of 9,400 U/mg and 7,900 U/ml, respectively; i.e, 89% of thetotal α-Gal A activity produced was secreted.

                  TABLE IV                                                        ______________________________________                                        Lysosomal Enzyme Activities Secreted In                                       Culture Media Of Transfected CHO Cells                                                      CHO Cell Line                                                                   DG44*     5-3.sub.250 *                                       Lysosomal Enzyme                                                                              Control   α-Gal A                                       ______________________________________                                        α-Galactosidase A                                                                       56        16,900                                              α-Arabinosidase                                                                         2.4       0.9                                                 α-Fucosidase                                                                            341       358                                                 β-Galactosidase                                                                          35.2      8.9                                                 β-Gaucuronidase                                                                          90.0      53.7                                                β-Hexosaminidase                                                                         2,290     2,090                                               α-Mannosidase                                                                           147       82.8                                                Acid Phosphatase                                                                              30.6      6.1                                                 ______________________________________                                         *Average of Triplicate Determinations in Two Independent Experiments.    

Since treatment of recombinant CHO cells with 50 mM butyrate has beenshown to specifically increase transcription of the stably integratedp91023(B) vector in CHO cells (Dorner, et al., 1989, J. Biol. Chem.264:20602; Andrews & Adamson, 1987, Nucl. Acids Res. 15:5461) anothertransfected clone, DGll, which was not amplified, was grown in thepresence of 5 mM butyrate (Table V). The intracellular levels of α-Gal Aactivity increased from 259 U/mg to 687 U/mg. Notably, in the presenceof butyrate, increased α-Gal A activity was secreted into the media (103to 675 U/ml), suggesting that secretion occured when the gene copynumber increased (or, more precisely, the steady state of α-Gal A mRNAwas increased). Incubation of butyrate-induced cells with 5 mMM-6-P (toprevent recapture of the secreted enzyme by the cell surface receptor)did not result a significant increase in the amount of α-Gal A secreted.

                  TABLE V                                                         ______________________________________                                        Butyrate Effect On α-Gal A Secretion in CHO DG11                                         α-Gal A Activity                                                          Cells    Media                                             Clone              (U/mg)   (U/ml)                                            ______________________________________                                        Control            259      102.6                                             Butyrate           687      675                                               Butyrate + 5 mM M-6-P                                                                            604      700                                               ______________________________________                                    

6.2.5. EFFECT OF SERUM CONCENTRATION ON SECRETION

To determine if the serum concentration of the growth media had aneffect on the levels of recombinant α-Gal A secretion, clone DG5.3 wasgrown in 100 mm culture dishes at a density of 5×10⁶ cells per dish, inthe presence of 0% to 10% dialyzed FCS for 5 days. There was no apparenteffect on α-Gal A secretion in cells grown with 2 5% to 10% serum (FIG.3). The decreased level of secretion by DG5.3 cells cultured in 0% and1% serum presumably reflected the poor growth of these cells.

6.2.6. PRODUCTION IN BIOREACTORS

To produce large quantities of recombinant human α-Gal A, 10⁸ cells ofclone DG5.3 which had been grown in the presence of 500 μM MTX(DG5.3₅₀₀), were used to seed a hollow fiber bioreactor. As shown inFIG. 4, the level of α-Gal A produced increased to about 10,000 U/ml perday. This level remained constant for about three months. In addition,the serum concentration required by these cells in the bioreactor wasstep-wise decreased to 1% without seriously decreasing α-Gal Aproduction (FIG. 4). A single 90-day run of this bioreactor resultedin >350 mg of active recombinant α-Gal A secreted into the culturemedia.

6.3. DISCUSSION

For human α-Gal A, post-translational modifications appear to beessential for stability and activity, as evidence by the fact that theunglycosylated enzyme expressed in E. coli was unstable andrapidly-degraded (Hantzopoulos & Calhoun, 1987, Gene 57:159). Inaddition, the α-Gal A subunit, which has four potential N-glycosylationsites, undergoes carbohydrate modification and phosphorylation forlysosomal delivery (Lemansky, et al., 1987, J. Biol. Chem. 262:2062).Previous characterization of α-Gal A purified from plasma and tissueidentified their different carbohydrate compositions, the plasmaglycoform having more sialic acid residues (Bishop, et al., 1978,Biochim. Biophys. Acta 524:109; Bishop, et al., 1980, Birth Defects16:1; p. 17; Bishop and Desnick, 1981, J. Biol. Chem. 256:1307).Moreover, clinical trials of enzyme therapy revealed that compared tothe tissue-derived form, the plasma glycoform had a prolonged retentionin the circulation and was more effective in depleting the circulatingaccumulated substrate following intravenous administration to patentswith Fabry disease (Desnick, et al., 1979, Proc. Natl. Acad. Sci. USA76:5326). Thus, the amplified expression of human α-Gal A in CHO cellswas chosen for the expression of this recombinant enzyme whose nativecomposition includes galactosyl and sialic acid residues (Ledonne, etal., 1983, Arch. Biochem. Biophys. 224:186).

Although this is the first human lysosomal hydrolase to be successfullyoverexpressed, an unexpected finding was the secretion of over 80% ofthe enzyme produced. This could result from several different mechanismsincluding (a) saturation of the mannose-6-phosphate receptor pathways;(b) a mutation that alters a critical glycosylation site; (c) failure toexpose the mannose-6-phosphate moiety for receptor binding; or (d) anunusually low affinity of recombinant α-Gal A for themannose-6-phosphate receptor (Reitman & Kornfeld, 1981, J. Biol Chem.256: 11977; Lang, et al., 1984, J. Biol. Chem. 259:14663; and, Gueze, etal., 1985, J. Cell. Biol. 101:2253; for review see, Kornfeld & Mellman,1989, Ann. Rev. Cell. Biol. 5:483). If the secretion of α-Gal A was dueto the saturation of the receptor-mediated pathway, then it would beexpected that the other endogenous lysosomal enzymes also would besecreted. However, the levels of secreted CHO hydrolases were unchanged,or decreased (Table IV). To rule out a possible mutation in the α-Gal AcDNA introduced during construction and integration of the vector(Calos, et al., 1983, Proc. Natl. Acad. Sci. USA 80: 3015), theintegrated vector DNA was amplified by the polymerase chain reaction.Ten subclones were completely sequenced in both orientations, and nomutations were identified. In companion studies of the purifiedrecombinant protein (described infra), it was shown that themannose-6-phosphate moiety was present on the enzyme and that the enzymebound efficiently to the immobilized mannose-6-phosphate receptor.Furthermore, to prove that the secretion of this protein in theexpression system utilized was not α-Gal A dependent, the cDNA encodinganother lysosomal hydrolase α-N-acetylgalactosaminidase, was insertedinto p91023(B) and amplified in CHO cells. Analogous to the observationswith α-Gal A, cells that were high expressors ofα-N-acetylgalactosaminidase (α-GalNAc), also secreted the recombinantenzyme in the medium.

The presence of functional mannose-6-phosphate moieties on the secretedenzyme implied that perhaps a different mechanism was responsible forits secretion. In fact, many other secreted proteins have been shown tocontain mannose-6-phosphate. Some of these proteins include lysosomalproteins while the location of others is not clear. These proteinsinclude, proliferin (Lee & Nathans, 1988, J. Biol. Chem. 263: 3521)secreted by proliferating mouse placental cell lines; epidermal growthfactor receptor in A-431 cells (Todderud & Carpenter, 1988, J. Biol.Chem. 263: 17893); transforming growth factor β1 (Purchio, et al., 1988,J. Biol. Chem. 263:14211); uteroferrin, an iron containing acidphosphatase secreted in large amounts by the uterine endometrium of pigs(Baumbach, et al., 1984, Proc. Natl. Acad. Sci. USA 81:2985); andcathepsin L (MEP), a mouse lysosomal cystein protease secreted by mouseNIH 3T3 fibroblasts (Sahagian & Gottesman, 1982, J. Biol. Chem. 257:11145). Of interest, transformation of NIH 3T3 cells with Kirstein virusresults in a 25-fold increase in the synthesis of MEP causing thisenzyme to be selectively secreted even though it contains functionalmannose-6-phosphate moieties (Sahagian & Gottesman, 1982, J. Biol. Chem.257:11145). Recently, the mechanism for the selective secretion of MEPhas been identified and it involves an inherent low affinity of MEP forthe mannose-6-phosphate receptor (Dong, et al., 1989, J. Biol. Chem.264: 7377).

It is also notable that the plasma-directed overexpression of yeastvacuolar carboxypeptidase Y in yeast results in over 50% of the normallyglycosylated protein secreted as the precursor form (Stevens, et al.,1986, J. Cell. Biol. 102:1551). Similar findings were observed for theyeast proteinase A gene (Rothman, et al., 1986, Proc. Natl. Acad. Sci.USA 83: 3248). Studies have suggested that the precursor glycoproteinshave subcellular localization signals located within the N-terminalpropeptide that are recognized by the secretion pathway, therebyprecluding delivery to the lysosome-like vacuole. It is notable that thesecretion of these yeast genes is gene-dosage dependent and that asimilar phenomenon is observed for the expression in CHO cells of humanα-Gal A. Also, it is of interest that the precursor form of the yeastenzymes was secreted. The plasma form of α-Gal A is more sialyated andsecreted, and others have shown that the lysosomal enzymes in humanurine are the precursor forms (Oude-Elferink, et al., 1984, Eur. J.Biochem. 139:489). However, N-terminal sequencing of recombinant α-Gal Aexpressed by DG44.5 revealed that the amino-terminus was identical tothat of α-Gal A purified from human lung (Bishop, et al., 1986, Proc.Natl. Acad. USA 83:4859). Thus, it is possible that the high-levelexpression of human lysosomal hydrolases results in their secretion dueto the inability to modify the precursor and/or inability of thesubcellular localization machinery to accommodate the intracellulardelivery of the overexpressed glycoprotein. However, this again wouldresult in the secretion of other lysosomal enzymes. Since no otherlysosomal enzymes are detected in the culture media, it is less likelythat secretion of α-Gal A results from saturation of a component of thesubcellular localization machinery.

Further studies, directed to determine amino acid, carbohydrate or otherdifferences (e.g., sulfation) between the secreted and intracellularforms of recombinant α-Gal A may provide insights into the mechanismunderlying the mislocalization and selective secretion of human α-Gal A.In addition, efforts to evaluate the generality of this observationshould include the overexpression of other human lysosomal enzymes. Thefact that large amounts of recombinant human α-Gal A are secreted by CHOcells permits the convenient production of the recombinant enzyme.Section 8, infra, describes a method for the purification of therecombinant enzyme and the characterization of its physical and kineticproperties including its receptor-mediated uptake by Fabry fibroblasts.

7. EXAMPLE: PURIFICATION, CHARACTERIZATION AND PROCESSING OF RECOMBINANTα-GalACTOSIDASE A

The subsections below describe the purification of human α-galactosidaseA cloned into the amplifiable eukaryotic expression vector, p91023(B),and overexpressed in Chinese hamster ovary (CHO) cells. The recombinantenzyme protein, was selectively secreted into the culture media and over200 mg was purified to homogeneity by a Fast Protein LiquidChromatographic procedure including affinity chromatography onα-galactosylamine-Sepharose. The purified secreted enzyme was ahomodimeric glycoprotein with native and subunit molecular weights ofabout 110 and 57 kDa, respectively. The recombinant enzyme had a pI of3.7, a pH optimum of 4.6, and a km of 1.9 mM toward4-methylumbelliferyl-α-D-galactopyranoside. It rapidly hydrolyzedpyrene-dodecanoyl-sphingosyl-trihexoside, a fluorescently labeledanalogue of the natural glycosphingolipid substrate, which was targetedwith apolipoprotein E to the lysosomes of the enzyme-producing CHOcells. Pulse-chase studies indicated that the recombinant enzyme assumedits disulfide-defined secondary structure in <3 min, was in the Golgi by5 min where it became Endo H resistant and was secreted into the mediaby 45-60 min. Both the intracellular and secreted forms werephosphorylated. The secreted enzyme subunit was slightly larger than theintracellular subunit. However, following endoglycosidase treatment,both subunits co-migrated on SDS-PAGE, indicating differences in theoligosaccharide moieties of the two forms. Treatment of the radiolabeledsecreted enzyme with various endoglycosidases revealed the presence ofthree N-linked oligosaccharide chains, two high-mannose types (Endo Hsensitive) and one complex type, the latter being Endo H and Fresistant. Analyses of the Endo H-released oligosaccharides revealedthat one had two phosphate residues which specifically bound toimmobilized mannose-6-phosphate receptors while the other was a hybridstructure containing sialic acid. These physical and kinetic propertiesand the presence of complex-type oligosaccharide chains on therecombinant secreted enzyme were similar to those of the native enzymepurified from human plasma. The secreted form of α-Gal A was taken up bycultured Fabry fibroblasts by a saturable process that was blocked inthe presence of 2 mMmannose-6-phosphate indicating that binding andinternalization were mediated by the mannose-6-phosphate receptor. Thebinding profiles of the recombinant secreted enzyme and the α-Gal Asecreted by NH₄ Cl-treated human fibroblasts to the immobilized receptorwere identical. The production of large amounts of soluble, activerecombinant α-Gal A in accordance with the invention, which is similarin structure to the native enzyme isolated from plasma, will permitfurther comparison to the native enzyme forms and the clinicalevaluation of enzyme replacement in Fabry disease.

7.1. MATERIALS AND METHODS 7.1.1. MATERIALS

Endo-β-N-acetylglucosaminidase H (Endo H),endo-β-N-acetylglucosaminidase D (Endo D), endoglycosidase F (Endo F)and peptide:N-glycosidase F (PNGase F) were obtained from BoeringerMannheim, Indianapolis, Ind. [³⁵ S]-methionine (>1,000 Ci/mmol),D-[2,6-³ H]-mannose (60 Ci/mmol), ³² P-Phosphorus (10 mCi/ml) andAmplify were obtained from Amersham, Arlington Heights, Ill. Pansorbinwas obtained fromCalbiochem, San Diego, Calif. 4-MU glycosides wereobtained from Genzyme, Cambridge, Ma. Freund's adjuvants, sphingomyelin(from brain) and phenylmethylsulfonyl fluoride (PMSF) were obtained fromSigma, St. Louis, Mo. QAE Sephadex, Sephadex G-25, octyl Sepharose andSuperose 6 were obtained from Pharmacia-LKB, Piscataway, N.J. The TLCsilica plates (cat. 5626) were purchased from EM Science, Gibbstown,N.J. The COS-1 cell line was obtained from the ATCC. All tissue culturereagents were obtained from Gibco, Grand Island, N.Y. Sinti Verse Iscintillation cocktail was obtained from Fisher, Pittsburgh, Pa. Theimmobilized mannose-6-phosphate receptor was obtained from Dr. StuartKornfeld, Washington University, St. Louis, Mo. Thepyrene-dodecanoyl-sphingosyl-trihexoside (P-C₁₂ STH) was obtained fromDr. Shimon Gatt, Hebrew University, Israel. Apolipoprotein E wasobtained from BTG Inc., Ness-Ziona, Israel.

7.1.2. CELL CULTURE

Cells were maintained at 37° C. in 5% CO₂ in Dulbecco's Modification ofEagle's Medium (DMEM) with 10% fetal calf serum (FCS) and antibiotics.The DG44 line was cultured in DMEM supplemented with HT (hypoxanthine,thymidine, Sigma) while the recombinant CHO line DG5.3 received DMEMsupplemented with 10% dialyzed FCS. .(Kaufman, et al., 1988, J. Biol.Chem. 263: 6352).

7.1.3. PURIFICATION OF RECOMBINANT α-Gal A

Recombinant CHO culture media was collected (20 L) and concentrated to500 ml using a Pellicon cassette tangential-flow concentrator, with amolecular weight cutoff of 10,000 daltons (Millipore, Mass). The pH ofthe concentrate was adjusted to 4.7 to 5.0 with 10 N HCl andsubsequently clarified by centrifugation at 10,000×g in an RC-5refrigerated centrifuge for 10 min.

All chromatographic steps were automated on an FPLC apparatus(Pharmacia) and were performed at room temperature. Approximately 100 mlof the media concentrate (˜20 mg of α-Gal A enzyme protein) was appliedto an α-Gal A affinity column (α-GalNH₂ Sepharose; 2.5×8 cm) (Bishop &Desnick, 1981, J. Biol. Chem. 256:1307) pre-equilibrated with buffer A(0.1M citrate-phosphate, pH 4.7, 0.15M NaCl). The column was washed withbuffer A until the protein concentration in the eluate returned to thepre-application level (˜200 ml) and was eluted with 150 ml of buffer B(0.1M citrate-phosphate, pH 6.0, 0.15M NaCl, 70.4 mM galactose). Theeluate was collected, concentrated to about 20 ml using anultrafiltration cell, molecular weight cutoff 30,000 daltons, underpositive nitrogen pressure (Amicon). The concentrate was mixed with anequal volume of buffer C (25 mM Bis-Tris, pH 6.0, 3M (NH₄)₂ SO₄),centrifuged at 10,000×g and the pellet which contained up to 40% of theactivity, was redissolved in buffer A and mixed with an equal volume ofbuffer C and centrifuged as above. The combined supernatants wereapplied to a column of Octyl-Sepharose (1.5×18 cm) pre-equilibrated withbuffer D (25 mM Bis-Tris, pH 6.0, 1.5M (NH₄)₂ SO₄). The column waswashed as above until the eluting protein concentration returned topre-application levels (˜100 ml) and the column was eluted with buffer E(5 mM sodium-phosphate, pH 6.0, 50% ethylene glycol). The product fromthree Octyl-Sepharose elutions, totalling approximately 75 ml, wasconcentrated as above to about 2 ml using an Amicon concentrator. Theconcentrate was finally applied to a column of Superose 6 (20-40 μm,Pharmacia, 1.6×100 cm) equilibrated in buffer F (25 mM sodium phosphate,pH 6.5, 0.1 M NaCl). The α-Gal A peak was collected, ˜20 ml,concentrated as above and stored in buffer F at 4° C.

7.1.4. ENZYME AND PROTEIN ASSAYS

α-Gal A was assayed using 4-methylumbelliferyl-α-D-galactopyranoside(4-MU-α-Gal) as previously described (Bishop, et al., 1980, In EnzymeTherapy in Genetic Diseases:2. Desnick, R. J. (Ed.). Alan R. Liss, Inc.New York, p. 17). Briefly, a stock solution of 5 mM 4-MU was prepared in0.1M citratephosphate buffer, pH 4.6 solubilized in an ultrasonic bath.The reaction mixtures containing 10-50 μl of enzyme preparation or cellextracts and 150 μl substrate, were incubated at 37° C. for 10-30 min.The reactions were terminated with the addition of 2.33 ml of 0.1Methylenediamine. One unit of activity is that amount of enzyme whichhydrolyzes 1 nmol of substrate/hour.

Endo H, Endo D, Endo F and PNGase F digestions were performed asdescribed (Tarentino, et al., 1989, Meth. Cell. Biol. 32:111). Sampleswere diluted to 0.2-0.5% SDS before digestion. All reaction volumes were50 μl. A drop of toluene was added to each reaction tube to preventbacterial growth. Briefly, Endo H digestions (5 mU/reaction) wereperformed at 37° C. overnight in 5 mM sodium citrate, pH 5.5 and 0.2 mMPMSF. Endo D digestions (10 mU/reaction) were performed at 37° C.overnight in 0.2M citrate phosphate buffer, pH 6.0 and 0.2 mM PMSF. EndoF digestions (50 mU/reaction) were performed overnight at 30° C. in 0.17M sodium acetate, pH 6.0, 1.6% NP-40 and 0.2 mM PMSF. PNGase Fdigestions (100 mU/reaction) were carried out overnight at 30° C. in0.17M potassium phosphate, pH 8.6, 1.6% NP-40, 0.2 mM PMSF.

Protein concentration was determined by the fluorescamine method(Bohlen, et al., 1973, Arch. Biochem. Biophys. 155:213) as modified byBishop et al. (Bishop et al., 1978, Biochim. Biophys. Acta 524: 109).

7.1.5. IN VIVO NATURAL SUBSTRATE ASSAY

For this assay, 30 nmoles of P-C₁₂ STH and 70 nmoles of-sphingomyelinwere mixed in a chloroform:methanol solution (1:1), evaporated undernitrogen and dried in a Speed-Vac (Savant). The pellet was resuspendedin 2 ml of saline, sonicated using a Heat Systems Ultrasonics, Inc.,Microson sonicator for 3-5 min at 40% output power and allowed to standat room temperature for 1 hour. Apolipoprotein E (80 μg) was added andthe mixture was incubated for an additional 15 min at room temperature.The liposomes were added to the culture media of recombinant CHO cellsand incubated at 37° C. in a CO₂ incubator for 1 to 4 hours. Cells wereremoved from the culture dishes by trypsinization, washed once in DMEMsupplemented with 10% fetal calf serum and twice with saline. The cellpellet was resuspended in chloroform-methanol and heated to 60° C. for10 min and centrifuged at 600×g for 10 min. The supernatant was driedunder nitrogen and the pellet resuspended in 100 μl ofchloroform:methanol. Samples were spotted on a silica gel thin layerchromatography plate and chromatographed in chloroform:methanol:water(90:10:1) for 45 min followed by chromatography inchloroform:methanol:water (75:25:4) for 30 min. Products were visualizedunder UV light (330 nm), excised from the plate by scraping, resuspendedin chloroform:methanol, and their fluorescence quantitated in a Farrandspectrofluoremeter (343 nm excitation, 378 nm emission).

7.1.6. POLYCLONAL ANTIBODIES

A New Zealand white rabbit (2 kg) was injected with 150 μg of purifiedsplenic α-Gal A in Freund's complete adjuvant prepared as follows: 150μg of α-Gal A was added to 0.5 ml of PBS in a glass syringe. Using astainless steel 21 guage needle, the PBS/α-Gal A solution was mixed with0.5 ml of Freund's complete adjuvant in a second glass syringe, until ahomogenous emulsion was obtained. The emulsion was injected into 8different subcutaneous sites (back) and 1 intramuscular site (thigh).Two months following the initial injection, the rabbit was boosted with50 μg of α-Gal A in Freund's incomplete adjuvant as above. Serum wascollected from an ear vein at days 8 and 12 following the boost. Thetiter was checked using a standard ELISA assay (Johnstone & Thorpe,1982, Iummunochemistry in Practice. Balckwell Scientific Publications,Oxford). Subsequent boosts were given approximately every two monthsfollowed by a bleeding 10 days later. A typical bleed yielded 30-40 mlof blood.

7.1.7. SDS-PAGE AND AUTORADIOGRAPHY

Polyacrylamide gel electrophoresis was carried out under reducingconditions (where appropriate) as described by Laemmli in a 1.5 mm thickslab containing 10% acrylamide (Laemmli, U.K., 1970, Nature 227:680).The gel was fixed in 10% acetic acid and 20% methanol for 30 min andthen soaked in Amplify for 30 min with agitation. Gels were vacuum driedfor 90 min (Hoffer) and exposed to Kodak X Omat AR for 4 to 72 hours.

7.1.8. ISOELECTRIC POINT AND pH OPTIMUM DETERMINATION

The isoelectric point was determined using QAE sephadex essentially asdescribed by Yang and Langer (Yang & Langer, 1987, Biotechniques5:1138). The pH optimum was determined in 25 mM sodium phosphate bufferat 37° C.

7.1.9. MANNOSE-6-PHOSPHATE RECEPTOR AFFINITY CHROMATOGRAPHY AND QAESEPHADEX CHROMATOGRAPHY

The 215 kDa mannose-6-phosphate receptor (M-6-P receptor) coupled toAffigel-10 was at a concentration of 0.4 mg/ml of packed gel. Samples,in binding buffer (50 mM imidazole, pH 7.0, 150 mM NaCl, 0.05% TritonX-100, 5 mM sodium-β-glycerolphosphate, 0.02% sodium azide), wereapplied to a 1.5×0.8 cm column at a flow rate of 0.3 ml/minute.Following sample application (5 ml), the column was washed with 5 ml ofbinding buffer and eluted with a nonlinear gradient ofmannose-6-phosphate in binding buffer (0-5 mM). This exponentialgradient (Dong, et al., 1990, J. Biol. Chem. 265:4210) was formed by anapparatus consisting of two chambers of 2.5 cm diameter and 1 cmdiameter. Fractions were collected (0.5 ml) and 10 μl aliquots assayedfor α-Gal A activity using 4-MU-α-Gal, and for radioactivity using 10 mlof Sinti Verse I scintillation coctail.

QAE Sephadex chromatography in a 3×0.8 cm column was performed asdescribed (Varki & Kornfeld, 1983, J. Biol. Chem. 258:2808; Varki &Kornfeld, 1980, J. Biol. Chem. 255:10847). Briefly, following digestionwith Endo H, the released oligosaccharides (labeled with [³ H]-mannose)were isolated and desalted on an 18×0.8 cm column of Sephadex G-25.Samples were applied to the column of QAE Sephadex and eluted withsuccessive 5 ml aliquots of 2 mM Tris, pH 8.0 containing 0, 20, 40, 80,100, 120, 140, 160, 200, 400 and 1,000 mMNaCl. Oligosaccharides elutedaccording to the number of their negative charges; 0 charge at 0 mMNaCl, 1 at 20 mM NaCl, 2 at 70 mM NaCl, 3 at 100 mM NaCl and 4 at 140 mMNaCl.

7.1.10. LABELING OF CELLS WITH [³⁵ S]-METHIONINE, [³ H]-MANNOSE AND [³²P]-PHOSPHOROUS

Confluent cultures in 100 mm dishes were washed once with 5 ml ofmethionine-free DMEM. A fresh aliquot of this medium (5 ml) was placedin each dish and cultures were incubated in a 37° C. incubator for 30min. The media was removed from the dishes and a fresh aliquot ofmethionine-free DMEM (1 ml), supplemented with 10% dialyzed FCS and50-100 μCi of [³⁵ S]-methionine was added. Cells were incubated at 37°C. for 3 to 5 min, the radioactive media was removed and cells washedtwice with DMEM plus FCS. Cells were chased for the indicated times in 5ml of DMEM plus FCS containing 2 mMmethionine. For overnight labeling,cultures received 5 ml of methionine-free DMEM supplemented withdialyzed FCS, glutamine, antibiotics, 10 mM NH₄ Cl and 200 μCi [³⁵S]-methionine.

For [³ H]-mannose labeling, cultures were grown as above in supplementedDMEM. Cells were washed with 5 ml of low-glucose DMEM and a freshaliquot of media was added. [³ H]-mannose (250 μCi; dried under nitrogenand resuspended in DMEM), was added and cells were incubated in a 37° C.incubator for 24 hours.

For ³² P labeling, cultures were switched to phosphate-free DMEMsupplemented with 10% dialyzed FCS. Following addition of [³²P]-orthophosphate (1 mCi) cultures were incubated in a 37° C., CO₂incubator for 24 hours.

7.1.11. CELL LYSIS AND IMMUNOPRECIPITATION

Cells grown in 100 mm culture dishes were washed twice with 5 ml ofphosphate buffered saline (PBS) and scraped into 12 ml conical tubesusing a rubber policeman and 10 ml of PBS. Following centrifugation at2,500 rpm for 10 min cells were resuspended in 1 ml of 25 mMNaPO₄, pH6.0 and received three 15-second bursts in a Branson cup sonicator. Celldebris was removed by centrifugation (10,000×g for 15 min at 4° C.).Alternatively, cells were washed as above and 1 ml of lysis buffer (50mM sodium phosphate, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.2 mMPMSF) was added to the dish. The culture dish was incubated at 4° C. for30 min and cells were transferred to a 1.5 ml microcentrifuge tube. Celldebris was removed as above.

Immunoprecipitation was carried out as described (Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual Cold Spring HarborLaboratory Press pp. 18.42-18.46). Briefly, 0.5 ml of cell lysate orculture media was placed in a 1.5 ml microcentrifuge tube and 50 μl ofpreimmune rabbit serum was added. The mixture was incubated at 4° C. for1 hour with gentle agitation. Fifty μl of Pansorbin was added andincubation was continued for 30 min. The mixture was clarified bycentrifigation at 10,000×g for 5 min, 100 μl of anti-α-Gal A polyclonalantibody was added and incubation was continued for 1 hour at 4° C. withgentle rocking. Pansorbin (100 μl) was added and incubation continuedfor 30 min as above. The tertiary S. aureus cells-antibody-antigencomplex was collected by centrifugation as above. The supernatant wasdiscarded and the pellet washed successively in NET buffer (50 mM sodiumphosphate, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% gelatin)supplemented with 0.5M NaCl, in NET buffer with 0.1% SDS and in TNbuffer (10 mM Tris, pH 7.5, 0.1% NP-40). The immunoprecipitated proteinwas denatured by heating at 100° C. for 5 min in the presence of 2% SDS,100 mM DTT (DTT was not used for experiments involving secondarystructure conformations). S. aureus cells were removed by centrifugationat 10,000×g for 5 min at room temperature.

7.2. RESULTS 7.2.1. PURIFICATION

Recombinant α-Gal A produced in the cell bioreactor was purified fromthe crude media by affinty chromatography on α-GalNH₂ -C₁₂ -Sepharose(Bishop & Desnick, 1981, J. Biol. Chem. 256:1307) followed byhydrophobic chromatography on Octyl Sepharose and gel filtration on a100 cm Superose 6 column as described above. Table VI shows a typicalpurification of a 20 mg lot of recombinant α-Gal A and the specificactivities of the enzyme at each stage of purification. The recombinantenzyme was essentially homogeneous, following the gel filtration step,and was >98% pure as judged by SDS-PAGE (FIG. 5). A minor contaminant ofbovine serum albumin was removed by an additional gel filtration step ona column of Blue-Sepharose (Travis, et al., 1976, Biochem. J. 157:301)resulting in an enzyme preparation which was greater than 99% pure asjudged by loading 20 μg of α-Gal A on SDS-PAGE.

                  TABLE VI                                                        ______________________________________                                        FPLC Purification Of Recombinant α-GAL A                                A Typical Purification Run Starting                                           With 20 MG Of α-GAL A                                                                                Fold     Yield                                   Step     U × 10.sup.3                                                                      U × 10.sup.3                                                                      Purification                                                                           %                                       ______________________________________                                        Media    39,750       5       1       100                                     GalNH.sub.2                                                                            36,500      680     136      91                                      Sepharose                                                                     Octyl    31,750    3,400     680      79                                      Sepharose                                                                     Superose 6                                                                             30,800    4,150     830      78                                      ______________________________________                                    

That the purification of recombinant α-Gal A would be facilitated bygrowth of the CHO cells in serum-free media was demonstrated bymetabolic labelling of total cellular and secreted protein. In contrastto the result seen in FIG. 5, radiolabeled α-Gal A was essentially theonly protein seen in the media of the high-expressor line, DG5.3 (FIG.6).

7.2.2. PHYSICOKINETIC PROPERTIES

Recombinant α-Gal A was found to have a subunit molecular weight of ˜57Kd based on SDS-PAGE (FIG. 5). The Km towards the artificial substrate4-MU-α-D-galactopyranoside was 1.9 mM (FIG. 7A) and the pH optimum andisoelectric point were 4.6 and 3.7 respectively (FIG. 7B and 7C).

In order to determine whether the recombinant enzyme recognized andhydrolyzed its natural substrate, liposomes containing thefluorescently-labeled α-Gal A substrate P-C₁₂ STH and apolipoprotein E(for lysosomal targeting) were incubated with CHO cells over-expressingα-Gal A (clone DG5.3). As shown in FIG. 8,recombinant lysosomal α-Gal Arapidly hydrolyzed the substrate to P-C₁₂ SDH (the dihexoside). Therapid hydrolysis of P-C₁₂ STH indicates that recombinant α-Gal A canrecognize this natural substrate analog and very efficiently hydrolyzeit. Also, since this substrate is targeted to the lysosome, cellassociated recombinant α-Gal A must be correctly targeted to thislocation. These results indicate that recombinant α-Gal A produced andsecreted by CHO cells is essentially identical to the enzyme purifiedfrom human plasma (Table VII).

                  TABLE VII                                                       ______________________________________                                        Property Comparison Of Recombinant α-GALACTOSIDASE A                    Enzyme Purified From Human Tissue                                                          α-Gal A                                                    Property       Spleen   Plasma   Recombinant                                  ______________________________________                                        MW-Subunit, (KDa)                                                                            53       57       57                                           pH Optimum     4.5      4.6      4.6                                          Isoelectric Point, PI                                                                        4.3      3.7      3.7                                          Km (4-MU-α-D-Gal), mM                                                                  2.5      1.9      1.9                                          Phosphorylation (M-6-P)                                                                      +        ?        +                                            Natural Substrate                                                                            +        +        +                                            Hydrolysis (GL-3)                                                             ______________________________________                                    

7.2.3. PROCESSING AND RATE OF SECRETION OF RECOMBINANT α-Gal A

Nascent polypeptides, transversing the endoplasmic reticulum assumesecondary structure conformations cotranslationally or soon after theirsynthesis is completed (Gething, et al., 1989, Meth. Cell. Biol.32:185). α-Gal A was labeled with [³⁵ S]-methionine for three min andthen chased with cold methionine for the indicated times.Immunoprecipitated α-Gal A was visualized on SDS-PAGE. The samples wereprepared without DTT in order to maintain disulfide bridges that mighthave formed during the chase, indicative of a secondary structureconformation. A control (+DTT) was prepared by boiling an aliquot of the60 minute sample in the presence of DTT to destroy disulfide bonds andthe secondary structure. At 0 min of chase (after 3 min of labeling)there was already a change in the mobility of this enzyme indicatingthat conformational changes occur cotranslationally or soon aftercompletion of synthesis (FIG. 9).

Arrival of the new polypeptide to the Golgi network was detected by theacquisition of Endo H resistant oligosaccharides (Gething, et al., 1989,Meth. Cell. Biol. 32:185). Radiolabeled α-Gal A (3 minute pulse) waschased with nonradioactive methionine and immunoprecipitated as above.The immunoprecipitates were then treated with Endo H and visualized onSDS-PAGE. Between 2 and 7 min of chase, the first Endo H-resistant formof α-Gal A could be detected, indicative of arrival of the recombinantenzyme at the Golgi, about 5 to 10 min following its synthesis (FIG.10). The majority of the Endo H sensitive form was rendered resistant by60 min of chase.

This enzyme transverses the Golgi network and is secreted at 45 to 60min of chase (FIG. 11). Analysis of total media, from [³⁵ S]-methioninelabeled cells, revealed that >95% of the secreted protein by therecombinant CHO cells was α-Gal A (FIG. 12).

7.2.4. ANALYSIS OF CARBOHYDRATE MOIETIES ON RECOMBINANT α-Gal A

There are four N-glycosylation consensus sequences (Asn-X-Ser/Thr) inthe α-Gal subunit predicted by the cDNA sequence. The fourth site isprobably not utilized since it contains a proline residue in the Xposition. Recombinant α-Gal A was digested with Endo H, Endo F, Endo Dand PNGase F. Digestion with PNGase F caused an ™7 kDa shift in mobilityon SDS-PAGE of half of the α-Gal A (FIG. 13). This change in molecularweight can be attributed to the removal of 3 N-linked carbohydratemoieties. Digestion of the recombinant enzyme with a cocktail of Endo H,Endo F and PNGase F did not result in any further decrease in molecularweight, indicating that all of the enzyme contains three N-linkedcarbohydrate moieties.

Endo D, a glycosidase with a strict specificity for the lower Manel-3branch of the high-mannose core pentasaccharide (Tarentino, et al.,1989, Meth. Cell. Biol. 32:111), did not have an effect on the mobilityof α-Gal A, indicating that the recombinant enzyme does not contain thistype of oligosaccharide (FIG. 13). Endo H and Endo F together resultedin a 4 kDa shift indicating that two out of the three oligosaccharideson this enzyme are of the high-mannose type (Varki & Kornfeld, 1980, J.Biol. Chem. 225:10847).

Interestingly, intracellular α-Gal A was completely sensitive to PNGaseF while half of the secreted enzyme was partially resistant to PNGase F(FIG. 14). Since this resistance was eliminated by co-treatment withEndo H and Endo F (FIG. 13), further studies are necessary with Endo Hand Endo F separately to determine the molecular nature of either theselective inhibition of PNGase F or the resistance of a proportion ofthe recombinant secreted enzyme to PNGase F digestion.

Having determined that the recombinant enzyme contains threeoligosaccharides, two of which are of the high-mannose type, the effectof inhibition of glycosylation was investigated (Furhmann, et al., 1985,Biochim. Biophys. Acta 825:95). Processing and secretion of recombinantα-Gal A is not affected by selective inhibition of oligosaccharideprocessing. In the presence of deoxynojirimycin (an inhibitor ofglucosidase I and II), deoxymannojirimycin (an inhibitor of mannosidaseI), and swainsonine (an inhibitor of mannosidase II) α-Gal A secretionrate remains the same as the controls (FIG. 15). However, tunicamycin(an inhibitor of oligosaccharide addition) inhibits secretion of α-Gal Aby as much as 80% (FIG. 15). The secreted enzyme fromtunicamycin-treated cultures could bind to a Con A Sepharose columnindicating that this enzyme is partially glycosylated, probably due toincomplete inhibition of glycosylation by tunicamycin. These resultsindicate that oligosaccharide addition but not the processing eventstested is necessary for maturation and secretion of the recombinantenzyme.

7.2.5. PHOSPORYLATION

Since the recombinant α-Gal A contained high-mannose moieties, therecombinant enzyme could contain M-6-P and be competent for receptormediated uptake. Cells from clone DG5.3 were metabolically labeled with[³² P]-orthophosphate for 12 hours and then the cell extracts and mediaimmunoprecipitated and visualized on SDS-PAGE. As shown in FIG. 16, bothcell-associated and secreted α-Gal A were phosphorylated, presumably attheir carbohydrate moieties as suggested by the in vitro experimentsdescribed above.

7.2.6. ANALYSIS OF ENDO H SENSITIVE OLIGOSACCHARIDES

The high-mannose oligosaccharides were removed by treatingimmunoprecipitated [³ H]-mannose labeled α-Gal A with Endo H. Theseoligosaccharides were analysed by chromatography on QAE Sephadex (Varki& Kornfeld, 1983, J. Biol. Chem. 258:2808; and, Varki & Kornfeld, 1980,J. Biol. Chem. 255:10847). Two major forms of these oligosaccharideswere detected, a form with 2 negative charges and one with 4 negativecharges (FIG. 17A). The negative charge can be contributed by aphosphodiester moiety (-1), a phosphomonoester moiety (-2) or sialicacid (-1). Treatment of these sugars with dilute HCl did not shift theprofile of any of the peaks indicating that there are no phosphodiestergroups on these sugars (FIG. 17B) (Varki & Kornfeld, 1983, J. Biol.Chem. 258:2808). Treatment with neuraminidase causes a shift of the -2peak resulting in two new peaks at 0 and -1 negative charges (FIG. 17°C.). Therefore, the charge of the -2 peak is contributed by sialic acid,most likely two moieties. The resulting -1 peak following neuraminidasetreatment is probably a partial digestion of the -2 peak by the enzyme.Treatment of these oligosaccharides with alkaline phosphatase caused ashift of the -4 peak to 0 negative charge (FIG. 17D). There was noeffect on the -2 peak, indicating that the charge of the -4 peak iscontributed by two phosphomonoester bonds while the -2 peak does notcontain any such bonds. Thus, it is evident from these results that EndoH releases two types of high-mannose oligosaccharides from recombinantα-Gal A, one containing sialic acid (possibly a hybrid oligosaccharide)and the other containing 2 phosphomonoester bonds (presumably asmannose-6-phosphate).

To further confirm these findings peaks -2 and -4 were chromatographedon an immobilized mannose-6-phosphate receptor column (FIG. 18).Although peak -4 interacted weakly with the receptor, it could be boundto the column and required the addition of mannose-6-phosphate forelution. A very weak interaction was observed between the receptorcolumn and the -2 peak, suggesting that a portion of these hybridoligosaccharides may contain M-6-P.

The weak interaction of the high-mannose oligosaccharides with the M-6-Preceptor could be explained by the absence of the protein core (Varki &Kornfeld, 1983, J. Biol. Chem. 258:2808). DG5.3 cells were labeled with[³⁵ S]-methionine and the secretions chromatographed on a column ofimmobilized mannose-6-phosphate receptor. Notably, the recombinantenzyme bound strongly to the column was eluted specifically by theaddition of 5 mMM-6-P (FIG. 19).

7.2.7. INTERACTION OF α-Gal A WITH THE MANNOSE-6-PHOSPHATE RECEPTOR

Since recombinant α-Gal A has been shown to contain mannose-6-phosphatemoieties, it was important to establish whether this was also true fornormal human α-Gal A. CHO proteins were labeled with [³⁵ S]-methioninein the presence of NH₄ Cl, to cause quantitative secretion of newlysynthesized lysosomal enzymes (Dean, et al., 1984, Biochem. J. 217:27).The media was collected and chromatographed on a column of immobilizedmannose-6-phosphate receptor. The column was eluted with a gradient ofmannose-6-phosphate as described above. This elution protocol canseparate lysosomal enzymes into low and high affinity receptor-bindingligands (Dong & Sahagian, 1990, J. Biol. Chem. 265:4210).

The recombinant enzyme co-eluted with the bulk of the lysosomal enzymesat an M-6-P concentration indicative of high affinity forms (FIG. 20A).The same experiment was performed with secretions of MS914 (normaldiploid human fibroblasts) cells (FIG. 20B) and 293 cells (humanadenovirus transformed embryonic kidney cells) (FIG. 20C). When the sameM-6-P gradient was applied, human α-Gal A also co-eluted with the bulkof the lysosomal enzymes, demonstrating that the recombinant enzymeexhibits affinity to the M-6-P receptor similar to that of the normalhuman enzyme.

7.2.8. RECEPTOR MEDIATED UPTAKE OF RECOMBINANT α-Gal A IN FABRYFIBROBLASTS

Fabry fibroblasts were incubated with varying amounts of the recombinantenzyme for 6 hours (FIG. 1). The enzyme uptake was saturatable and wasspecifically inhibited by the addition of 2 mMM-6-P in the uptake media,indicating that the uptake was via the cell surface M-6-P receptor.

8. EXAMPLE: α-Gal A-PROTEIN A FUSION EXPRESSED IN MAMMALIAN CELLS

The subsections below describe a fusion construct of the humanα-Galactosidase A cDNA and the staphylococcal protein A IgG bindingdomain E expressed in COS-1 cells and purified to apparent homogeneityby IgG affinity chromatography. The fusion construct was engineeredusing PCR techniques to insert the 16 nucleotide collagenase cleavagerecognition sequence between the α-Gal A and the protein A domain Esequence. In addition, the termination codon was deleted from the α-GalcDNA and inserted at the terminus of the domain E sequence. Transientexpression of the fusion construct in COS-1 cells resulted in a 6 to7-fold increase over endogenous levels of α-Gal A activity andsignificant secretion into the media (4,000 units; nmoles/hour). Thefusion protein from the culture media was purified to homogeneity on IgGsepharose chromatography. After collagenase treatment, the liberatedα-Gal A was separated from the protein A peptide by IgG chromatography.By this method over 85% of secreted α-Gal A fusion protein was purifiedas the active, glycosylated homodimeric protein. This method should beuseful for the expression and rapid purification of normal and mutantproteins. In addition, this construct has been inserted into the CHODG44 cells so that large amounts of the secreted recombinant enzyme canbe produced and rapidly and efficiently purified.

8.1. MATERIALS AND METHODS 8.1.1. MATERIALS

Restriction endonucleases, Taq polymerase, T4 ligase and pGem plasmidswere obtained from Promega (Madison, Wis.). Vector pRIT5 andIgG-Sepharose were purchased from Pharmacia (Piscataway, N.J.).Sequenase sequencing kits were from United States Biochemical Corp.(Cleveland, Ohio). Collagenase was obtained from Sigma (St. Louis, Mo.).Oligonucleotides were synthesized using an Applied Biosystems DNAsynthesizer model 380B.

8.1.2. CELL CULTURE AND TRANSFECTIONS

COS-1 cells were obtained from the ATCC (Rockville, Md.). The cells werecultured by standard techniques in Dulbecco's Modified Eagle's Medium(DMEM) with 10% fetal calf serum and antibiotics.

Exponentially growing COS-1 cells (5×10⁶ cells /T75 flask) were detachedfrom the plastic by trypsinization, collected by centrifigation at3,000×g, and then washed once in ice-cold electroporation buffer(phosphate buffered sucrose: 272 mM sucrose, 7 mM sodium phosphate, pH7.4, containing 1 mMMgCl₂). Following centrifugation at 3,000×g, thecells were resuspended in 0.8 ml of electroporation buffer and placed inan electroporation cuvette with a 0.4 cm gap. Ten to fifteen μg ofplasmid DNA was added and cells were kept on ice for 5 min. Thecell-containing cuvette was placed in a Gene Pulser electroporationapparatus (Bio-Rad) and the cells were pulsed at 350 V, 25 μF. The cellswere maintained on ice for an additional 10 min and then placed into a100 mm culture dish containing 10 ml of growth medium.

8.1.3. PCR, DNA SEQUENCING AND VECTOR CONSTRUCTIONS

The fusion construct was synthesized using a recently described PCRtechnique (Ho, et al., 1989, Gene 77:51; Kadowaki, et al., 1989, Gene76:161). Briefly, the full-length α-Gal A cDNA was subcloned into thepGEM plasmid and the resulting pG6-AGA plasmid was used for PCRamplification of the α-Gal A sequence with primers designed to deletethe termination codon, to add a collagenase cleavage consensus sequenceat the 3' end and to include an Eco RI recognition sequence at the 5'end of the cDNA (FIG. 22). The sense primer was5'-CCGAATTCATGCTGTCCGGTCACCGTG-3' [SEQ ID NO:10] and the antisenseprimer was 5'-CGCCGGACCAGCCGGAAGTAAGTCTTTTAATG-3'[SEQ ID NO:11]. Theprotein A domain E (Nilsson, et al., 1985, EMBO J. 4:1075) was similarlyamplified with the collagenase consensus sequence in the 5'oligonucleotide; the sense and antisense oligonucleotides were5'-CCGGCTGGTCCGGCGCAACACGATGAAGCT-3' [SEQ ID NO:12] and5'GGCCGAATTCCGGGATCCTTATTTTGGAGCTTGAGA-3'[SEQ ID NO:13], respectively.The 1323 nt and 201 nt products of the α-Gal A and protein A PCRreactions were gel-purified on an 0.8% agarose gel and mixed togetherfor the fusion PCR reaction. The sense primer from the α-Gal A reactionand the antisense primer from the protein A reaction were used for thefinal fusion reaction. The product of this reaction was digested withEco RI and ligated into the Eco RI digested plasmid pGEM 4Z. The proteinA domain E and junctions between the linker and α-Gal A and protein Awere confirmed by the dideoxynucleotide chain termination sequencingmethod of Sanger (Hanahan & Meselson, 1985, Methods Enzymol. 100:333).The confirmed fusion sequence then was digested with Eco RI andsubcloned into the eukaryotic expression vector p91023(B).

8.2. RESULTS 8.2.1. CONSTRUCTION OF α-Gal A-PROTEIN A (AGA-PA) FUSION

FIG. 22 shows the strategy used for the construction of the α-GalA-Protein A domain E fusion sequence. The full-length α-Gal A cDNA (1323nt) and protein A domain E sequence (201 nt) were amplified separatelyand then fused by a second PCR amplification (FIG. 22) using the 5'α-Gal A cDNA sense primer (P1) and the 3' Protein A antisense primer(P4). The primers were designed to (a) eliminate the α-Gal A TAA stopcodon; (b) insert the 16 nt collagenase cleavage consensus recognitionsequence encoding Pro Ala Gly Pro between the α-Gal A and protein A cDNAsequence; and (c) introduce a TAA stop codon at the 3' end of protein Adomain E. The integrity of this construct was confirmed by sequencingthe protein A domain, linker and 3' of the α-Gal A cDNA (FIG. 23).

8.2.2. EXPRESSION OF pAGA-PA IN COS-1 CELLS

Seventy-two hours after transfection with the pAGA-PA construct, maximallevels of 4MU α-Gal activity were detected in cell extracts and in thespent culture media (Table VIII).

                  TABLE VIII                                                      ______________________________________                                        Transient Expression Of AGA-PA Construct                                      In COS-1 Cells. Following Transfection A                                      7-Fold Increase In Endogenous α-Gal A Activity                          Was Observed. Also, An Increase Of α-Gal A In                           The Culture Media Was Observed                                                               α-Gal A Activity*                                                         Cells    Media                                               COS-1 CELLS      (U/mg)   (U/ml)                                              ______________________________________                                        Control            210     0                                                  Transfected      1,300    400                                                 ______________________________________                                         *Assayed using 4MUα-Gal as substrate.                              

Compared to the endogenous α-Gal A activity in COS-1 cells of 210 U/mg,the transfected cells expressed 1300 U/mg. No α-Gal A activity wasdetected in the spent culture medium of uninfected COS-1 cells whereas72 hours after transfection, 400 units of activity were secreted intothe media.

8.2.3. AFFINITY PURIFICATION OF α-Gal A

The spent media from a single 100 mm dish of COS-cells was collected 72hours after transection and applied to a column of IgG-Sepharose.Minimal activity of α-Gal A passed through the column during sampleapplication (flow-through), or during the buffer wash (Table IX).However, more than 95% of the bound α-Gal A fusion protein was eluted bythe addition of 0.5M acetic acid (elution buffer).

                  TABLE IX                                                        ______________________________________                                        IgG Sepharose Chromatography Of The α-Gal A                             Protein A Fusion Product From The Culture                                     Media Of Transfected COS-1 Cells                                              Purification  α-Gal A Activity*                                         Step          (U/ml)                                                          ______________________________________                                        Medium        4,400                                                           Flow-Through    10                                                            Buffer Wash      0                                                            Elution**     4,200                                                           ______________________________________                                         *Ten ml of culture media were applied to the column, washed and eluted as     described in "Methods". α-Gal A activity was assayed using              4MUα-Gal as substrate.                                                  **0.5 M HAc, pH 3.4                                                      

8.2.4. RELEASE OF THE PROTEIN A DOMAIN FROM THE AGA-PA FUSION PROTEIN

The affinity purified fusion protein was treated with collagenase for 1hour and the reaction products were rechromatographed on the IgGaffinity column (Table X). The Protein A domain E was readily bound tothe IgG column, whereas the human α-Gal A was eluted in theflow-through. Almost 90% of the applied activity was eluted. Based onthe specific activity of the purified enzyme, it was estimated that thisprocedure resulted in 90% pure enzyme.

                  TABLE X                                                         ______________________________________                                        Treatment Of α-Gal A-Protein Fusion With                                Collagenase. Upon Treatment The Bindng of                                     The IgG Column Decreased From 69% To 11%.                                                  % Of Recovered α-Gal A Activity*                                        COLLAGENASE**                                                    STEP         -            +                                                   ______________________________________                                        Flow-Through 31           89                                                  Elution      69           11                                                  ______________________________________                                         *Assayed using 4MUα-Gal as substrate; a total of 4,200 units of         α-Gal A activity was applied.                                           **Collagenase treatment for 1 hour at 25° C.                      

9. EXAMPLE: IN VIVO MODIFICATION OF RECOMBINANT HUMAN α-Gal AGLYCOSYLATION BY α 2, 6-SIALYLTRANSFERASE

The example presented here describes a method whereby recombinant humanα-Gal A enzyme is produced in a form that more closely resembles thenative plasma glycoform of the enzyme. Specifically, human α-Gal A isproduced in CHO cell lines that have been engineered to contain anα-2,6-sialyltransferase gene such that the α-Gal A protein made in thesecell lines is sialylated. Results presented below demonstrate that thebiologically active α-Gal A enzyme produced using such cell linesexhibit a broader tissue distribution, which can enhance thisrecombinant α-Gal A's therapeutic value.

9.1. MATERIALS AND METHODS 9.1.1. CONSTRUCTION OF THEα2,6-SIALYLTRANSFERASE EXPRESSION VECTOR, pST26

The 1.6 kb rat CDNA (ST3) encoding the complete amino acid sequence ofthe β-galactoside α2,6-sialyltransferase (Gal α2,6ST; Weinstein et al.,1987, J. Biol. Chem. 262: 17735) was subcloned into the Bam HI site ofthe mammalian expression vector pRLDN, a gift from Smith, Kline andFrench Laboratories, resulting in the vector designated pST26. Thisconstruct was introduced by electroporation into the CHO cell lineDG5.3-1000Mx, a high overexpressor of human α-galactosidase A. Cloneswere selected by growth in media containing 500 μg/ml of G418 to selectfor expression of the pST26 neogene. Positive clones were individuallyisolated using cloning rings and then each was analyzed for expressionof total secreted recombinant human α-galactosidase A. In addition, thepercent of the secreted recombinant α-galactosidas A that containedα2,6-sialic acid residues was determined by Sambucus nigra agglutinin(SNA) chromatography on a column of SNA-Sepharose as described by Lee etal. (1989, J. Biol. Chem. 264:13848-13855).

9.1.2. SNA-LECTIN FLUORESCENCE MICROSCOPY

For fluorescence microscopy, positive DG5.3-1000Mx-ST26 clones weregrown on 12-mm glass coverslips and then stained with fluoresceinisothiocyanate (FITC)-tagged SNA as previously described (Lee et al.,1989, J. Biol. Chem. 264:13848-13855). Briefly, the cells were fixedwith a fresh solution of 2% para-formaldehyde, 0.1% glutaraldehyde inphosphate buffered saline (PBS), pH 7.0, for 1 hr at 23° C., andsubsequently blocked with 50 mM NH₄ Cl in PBS for 30 min at 23° C. Cellswere incubated with FITC-SNA at a concentration of 25 mg/ml in PBS for30 min at 23° C. Following washing in PBS, the coverslips were mountedin 15% vinol 205 polyvinol alcohol, 33% glycerol, 0.1% sodium azide in100 mM Tris, pH 8.5. The cells were viewed with a Zeiss Photomat IIIfluorescence microscope and were photographed using Kodak Gold colorfilm, ASA 25.

9.1.3. PURIFICATION OF RADIOLABELLED HUMAN α-GAL A WITH AND WITHOUTα2,6-SIALIC ACID RESIDUES

Parental DG5.3-1000Mx cells and modified DG5.3-ST26.1 cells were grownin 1 liter roller bottles (-5×10⁸ cells) with 125 ml of DMEM, containing10% dialyzed fetal calf serum (GIBCO, Grand Island, N.Y.) and 500 μCi[³⁵S]-methionine. The radioactive medium was replaced daily for three days,and the spent medium from each line was collected, pooled andconcentrated using a stirred cell concentrator (Amicon, Beverly, Mass.).Approximately 2 mg (4×10⁶ U) of recombinant enzyme secreted from theDG5.3-1000Mx and DG5.31000Mx-ST26.1 cell lines, respectively, wasindividually purified to a specific activity of 100 cpm/U of enzyme. Thepurified, radiolabeled, secreted recombinant α-galactosidase Apreparations (0.5 mg each) were treated individually with 5 units ofacid phosphatase (Sigma) and the dephosphorylated forms also were usedfor intravenous injections.

9.1.4. IN VIVO HALF-LIFE AND TISSUE DISTRIBUTION OF RECOMBINANT α-GAL A

For the plasma half-life determinations, each radiolabelled α-Gal A formwas injected into the tail vein of two CDI female mice. Each animal wasbled through the sinus plexus at timed intervals over a 10-20 minperiod. Blood samples were centrifuged in an IEC Microhematocritcentrifuge and 50 μl of plasma was added to an aqueous scintillationfluid (AquaSol, NEN, Boston, Mass.) for counting. To assess the tissuedistribution of the different enzyme forms, two mice for each enzymewere sacrificed by decapitation 4 hr after enzyme injection, selectedtissues were removed, and the radioactivity in various tissues wasdetermined and expressed per gram of tissue wet weight. Each value isthe average of two independent experiments.

9.2. RESULTS 9.2.1. INTRODUCTION OF PST26 INTO DG5.3-1000Mx CHO CELLSAND DEMONSTRATION OF α2,6-SIALYLTRANSFERASE ACTIVITY IN G418-SELECTEDCLONES

Following electroporation of the pST26 plasmid into CHO DG5.3-1000MXcells, transformed cells were selected which were resistant to theantibiotic G418. From the pool of resistant cells, 10 individual cloneswere isolated and expanded for further studies. These clones weredesignated DG5.3-1000Mx-ST26.1 to ST26.10. As evidence for the presenceof α2,6-sialyltransferase activity in these cells, each cell line wasstained with FITC-SNA and then examined by phase contrast andfluorescence microscopy. All ten of the DG5.3-1000Mx-ST26 cell lineswere positively stained by the fluorescent lectin, indicating thatvarious cell membrane glycoproteins served as acceptors for theexpressed α2,6-sialyltransferase. In contrast, the FITC-lectin did notstain the parental DG5.3-1000Mx cells.

9.2.2. CHARACTERIZATION OF RECOMBINANT HUMAN SECRETED α2,6-SIALYLATEDα-GAL A

Each of the ten DG5.3-1000Mx-ST26 cell lines and the parentalDG5.3-1000Mx cells were grown in 100 mm dishes at the same cell density.The amount of secreted human α-Gal A activity per mg protein of totalcultured cells was determined for each cell line. The DG5.3-1000Mx-ST26lines secreted from 28 to 100% of the α-Gal A secreted by the parentalcell line (16,000 U/mg protein, Table XI).

To determine the percent of the recombinant human secreted α-Gal A thatcontained α2,6-sialic acid residues, the secreted enzyme waschromatographed on the immobilized SNA column, and the bound enzyme waseluted with 0.4M lactose. As shown in Table XI, the individual cloneshad 55 to 100% of the applied enzymatic activity that was specificallybound and eluted from the lectin column. Notably, cloneDG5.3-1000Mx-ST.1 produced the highest amount of recombinant α-Gal A,essentially all the enzyme form was sialylated and bound the immobilizedSNA column. In contrast, recombinant α-Gal A secreted by the parentalDG5.3-1000MX cells had little, if any binding to α2,6-sialicacid-specific lectin (Table XI).

                  TABLE XI                                                        ______________________________________                                        DG5.3-1000Mx-ST26 CHO Cell Lines                                              Secreting Human α2,6-Sialylated α-Galactosidase A                            Percent of     α-Gal A                                       Clone/     Secreted α-Gal A                                                                       Secretion                                           Subclone   Bound to SNA-Lectin                                                                          (% DG5.3-1000Mx*)                                   ______________________________________                                        DG5.3-1000MX                                                                              5             100                                                 DG5.3-1000Mx-                                                                 ST26 Subclones:                                                               1          100            100                                                 2          99             76                                                  3          93             72                                                  4          79             70                                                  5          74             64                                                  6          73             85                                                  7          72             85                                                  8          55             84                                                  9          51             81                                                  10         28             79                                                  ______________________________________                                         *Activity is expressed as units per mg of cellular protein; The DG5.3         1000Mx line secreted .sup.˜16,000 U/mg.                            

9.2.3. PLASMA HALF-LIFE AND TISSUE DISTRIBUTION OF α2,6-SIALYLATED HUMANα-GAL A IN MICE

To determine if the in vivo clearance of recombinant humanα2,6-sialylated α-Gal A was different than that of thenon-α2,6-sialylated enzyme, mice were injected with each form and theircirculating half-lives were determined. As shown in FIG. 24A, thepresence of α2,6-sialic acid moieties increased the half-life of theenzyme in the circulation from 14 to almost 30 min. Further, treatmentof the α2,6-sialylated and non-α2,6-sialylated recombinant enzymes withacid phosphatase increased the in vivo plasma half-life of thenon-α2,6-sialylated enzyme (T_(1/2) from 14 to 24 min), whereas that ofthe phosphatase-treated e2,6-sialylated enzyme remained the same atabout 28 min (FIG. 24B).

As depicted in FIG. 25, the presence or absence of α2,6-sialic acidmoieties and/or phosphate residues, on the secreted recombinant humanα-Gal A forms had a marked effect on their respective tissuedistributions following intravenous administration to mice. A greaterpercentage of the total α2,6-sialylated α-Gal A injected was recoveredin the lungs, kidney and heart, as compared to the non-α2,6-sialylatedenzyme; in contrast, less of the α2,6-sialylated enzyme was recovered inthe spleen. Interestingly, when the α2,6-sialylated enzyme was treatedwith acid phosphatase, the modified enzyme was redistributed to theliver and spleen, presumably to their reticuloendothelial cells (FIG.25). Acid phosphatase treatment of the non-α2,6-sialylated enzymeresulted in the recovery of significantly more enzyme than itsnon-phosphatase treated form in the lungs and heart whereas, less wasrecovered in the kidney (FIG. 25).

10. EXAMPLE: OVEREXPRESSION OF HUMAN LYSOSOMAL PROTEINS RESULTS IN THEIRINTRACELLULAR AGGREGATION, CRYSTALLIZATION IN LYSOSOMES, AND SELECTIVESECRETION

The example presented here describes a method, which is an inherentfeature of the invention and was first noted while overexpressing α-GalA, whereby certain proteins which are normally intracellularly targetedmay be recombinantly expressed in a secreted form. Using severalrecombinant lysosomal enzymes, the results demonstrate thatoverexpression of such proteins in CHO cells presumably leads toaggregation which, in turn, causes the protein to be secreted, ratherthan being targeted to the intracellular vesicles, in this case,lysosomes, to which they would normally be sent. This surprisingsecretion feature facilitates easy purification of the recombinantprotein produced.

10.1. MATERIALS AND METHODS 10.1.1. CONSTRUCTION OF PLASMIDS FORLYSOSOMAL ENZYME OVERPRODUCTION

Construction of the α-Gal A expression construct, p91-AGA, has beendescribed supra (Section 6.1.2). The analogous expression constructs forhuman acetylgalactosaminidase (designated p91-AGB) and acidsphingomyelinase (designated p91-ASM) and the respective transientexpression of each in COS-1 cells have been described (Wang et al.,1990, J. Biol. Chem. 265:21859-21866; Schuchman et al., 1991, J. Biol.Chem. 266:8535-8539).

10.1.2. CELL CULTURE, ELECTROTRANSFECTION, AND GENE AMPLIFICATION

CHO cells were grown and maintained as described supra, in Section6.1.3. Likewise, electroporation was performed as described supra, inSection 6.1.3.

Butyrate stimulation of the α-Gal A expressing CHO cells was performedas previously described (Dorner et al., 1989 J. Biol. Chem.264:20602-20607). Briefly, cells were plated in 100 mm dishes andallowed to grow in 10 ml of DMEM supplemented with 10% dFCS for 2 days.The media was removed and replaced with 10 ml of DMEM supplemented with10% dFCS containing 5 mM sodium butyrate. Cells were incubated for 16 hrat 37 ° C. in a CO₂ incubator and then cells and culture media wereharvested and the α-Gal A activity was determined.

10.1.3. ULTRASTRUCTURAL AND IMMUNOLABELING STUDIES

DG5.3-1000Mx cells were grown to confluency in 100 mm dishes. Followingtrypsinization (0.25% trypsin, EDTA), they were washed twice inphosphate buffered saline (PBS) and pelleted at 1,500 g for 5 min atroom temperature. Cells were then fixed for 1 hr with 3% glutaraldehydein PBS, followed by fixation in PBS-buffered 1% OsO₄ for 30 min at roomtemperature. Samples were then dehydrated with graded steps of ethanol,infiltrated with propylene oxide and embedded in Embed 812 (ElectronMicroscopy Sciences, Fort Washington, Pa.). 1 μm sections were cut fromrepresentative areas. Ultrathin sections were prepared and stained withuranyl acetate and lead citrate, and then were viewed with an electronmicroscope (JEM 100 CX, Jeol. USA, Peabody, Ma.).

For immunodetection, sections were prepared as above, and afterembedding, they were mounted on Formvar-coated nickel grids (FormvarScientific, Marietta, Ohio), incubated with goat serum in PBS for 30 minat 37° C. to block nonspecific binding, washed six times with PBS andthen incubated with affinity-purified rabbit anti-α-Gal A antibodies for1 hr. The sections were washed extensively as above and then wereincubated with 10 nm gold particles conjugated to protein A (AmershamCorp., Arlington, Ill.) for 1 hr at 37° C. After washing with PBS,sections were fixed with 3% glutaraldehyde in PBS for 15 min at roomtemperature, washed again with PBS and then examined under the electronmicroscope.

10.1.4. SDS-PAGE AND AUTORADIOGRAPHY

PAGE gel electrophoresis was carried out under reducing conditions asdescribed by Laemmli (1970 Nature (London) 277:680-685) in 1.5 mm thickslab gels containing 10% acrylamide. The gel was fixed in 10% aceticacid and 20% methanol for 30 min and then soaked in Amplify (AmershamCorp.) for 30 min with agitation. Gels were vacuum dried for 90 min(Hoffer Scientific Instruments, San Francisco, Calif.) and thenautoradiographed with Kodak X Omat AR film (Eastman Kodak Co.) for 4-24hrs.

10.1.5. IN VITRO STUDIES OF α-GAL A AGGREGATION

The possible formation of insoluble α-Gal A aggregates at varying enzymeand hydrogen ion concentrations was investigated. Using a stock solutionof purified, secreted α-Gal A (16 mg/ml in 10 mM Tris buffer, pH 7.0),appropriate aliquots were placed in glass borosilicate tubes and thevolumes were brought to 200 μl with distilled water so that with theaddition of 100 μl of the appropriate buffer (0.5M2-(N-morpholino)ethanesulfonic acid, at pH 5.0, 5.5, 6.0, 6.5 or 7.0),the final α-Gal A concentrations (0.1-10 mg/ml) would be achieved atspecific pH values. After incubation for 10 min at room temperature, theturbidity of each solution was determined by measuring the OD at 650 nmin a spectrophotometer (Spectronic 1201, Milton Roy Co., Rochester,N.Y.) using a 1-cm path cuvette. As a control, 1 mg/ml of purified,secreted α-Gal A was mixed with increasing BSA concentrations (0.1-10mg/ml) and the turbidity of each solution was determined. Similarexperiments were performed with solutions of α-Gal A (10 mg/ml) and BSA(2 mg/ml), at decreasing pH (from pH 7.0 to 5.0). After incubation andcentrifugation as above, the supernatants and pellets were subjected toSDS-PAGE.

10.1.6. ENZYME AND PROTEIN ASSAYS

The α-Gal A activities in the cell lysates and media were determinedusing 5 mM 4-methylumbelliferyl-α-D-galactopyranoside (4MU-α-Gal)(Genzyme Corp., Cambridge, Ma.) as previously described (Bishop andDesnick, 1981 J. Biol. Chem. 256:1307-1316). Briefly, a stock solutionof 5 mM 4MU-α-Gal was prepared in 0.1 M citrate/0.2M phosphate buffer,pH 4.6, in an ultrasonic bath. The reaction mixture, containing 10-50 μlof cell extract and 150 μl of the stock substrate solution, wasincubated at 37° C. for 10 to 30 min. The reaction was terminated withthe addition of 2.3 ml of 0.1M ethylenediamine. The fluorescence wasdetermined using a Ratio-2 System Fluorometer (Optical TechnologyDevices, Elmsford, N.Y.). 1U of activity is the amount of enzyme thathydrolyzed one nmol of substrate per hour. The activities ofα-mannosidase, β-galactosidase, β-hexosaminidase, β-glucuronidase, acidphosphatase and α-N-acetylgalactosaminidase were measured using theappropriate 4-methylumbelliferyl substrate. The activity of acidsphingomyelinase was determined according to Gal et al. (1975, N. Eng.J. Med. 293:632-636). Protein concentrations were quantitated by thefluorescamine method (Bohlen et al., 1973, Arch. Biochem. Biophys.155:213-220) as modified by Bishop et al. (1978, Biochem. Biophys. Acta524:109-120).

10.2. RESULTS 10.2.1. OVEREXPRESSION RESULTS IN CRYSTALLINE STRUCTURESCONTAINING HUMAN α-GAL A IN MEMBRANE-LIMITED VESICLES

Ultrastructural examination of the stably amplified DG5.3-1000Mx cellsrevealed numerous 0.25 to 1.5 μm crystalline bodies which had orderedtriangular lattices in membrane-limited vesicles throughout thecytoplasm (FIG. 26A and 26B). the repeat within these crystallinestructures was about 20 nm. These structures were particularly abundantin lysosomes (FIG. 28A) and in vesicles which appeared to be dilated TGN(FIG. 28B) (Hand and Oliver, 1984, J. Histochem. Cytochem.,42:403-442;Griffith and Simons, 1986, Science 234:438-443; McCracken, 1991, inIntracellular Traficking of Proteins, Steer and Hanover, eds., CambridgeUniversity Press, New York pp. 461-485). Of note, normal Golgistructures were not observed in these cells, whereas Golgi complexeswere readily identified in the parental DG44 cells (FIG. 26E, inset).When osmium-glutaraldehyde fixed sections of the DG5.3-1000Mx cells wereincubated with affinity-purified rabbit anti-human α-Gal A antibodiesand then with protein A-conjugated gold, these crystalline structureswere specifically stained by gold particles (FIG. 26C and 26D). That thecrystalline structures were immunogold labeled, even though the sectionswere fixed in osmiumglutaraldehyde, suggested that these structures wereprimarily, if not solely, composed of the human enzyme.

To determine whether the crystalline structures were present in clonesexpressing lower levels of α-Gal A, clones DG5.3-0Mx, -1.3Mx, -250Mx,and -1000Mx were grown to confluency and examined by electronmicroscopy. Although the TGN was increasingly dilated with increasingα-Gal A expression, only the DG5.3-1000Mx clone contained crystallinearrays in lysosomes. Similarly, clone DG5.3-1.3Mx was stimulated with 5mM sodium butyrate for 18 hrs to increase transcription of theintegrated vector containing the α-Gal A cDNA and then examinedultrastructurally. Compared to the untreated clone, butyrate treatmentresulted in the presence of dilated organelles including manymembrane-bound structures containing dense material. These resultsindicated that crystal formation was α-Gal A concentration dependent.Furthermore, to assess whether crystal formation was specific to α-GalA, clone AGB14.8-1000Mx, which overexpressesα-N-Acetylgalactosaminidase, was examined. No crystalline arrays wereobserved, but numerous dilated structures were seen similar to those inthe DG5.3-1.3Mx clone, suggesting that expression of the recombinantacetylgalactosaminidase had not reached the critical level necessary forcrystal formation.

10.2.2. α-GAL A AND α-N-ACETYLGALACTOSAMINIDASE AGGREGATE AT HIGHCONCENTRATION AND LOW pH

Since it was conceivable that the overexpression of α-Gal A resulted inthe formation of soluble and particulate aggregates that did not bind toor were inefficiently bound by the M6PR and/or the sulfotransferase inthe TGN, the possible aggregation of α-Gal A was assessed in vitro atvarying enzyme and hydrogen ion concentrations. As shown in FIG. 27A,the amount of α-Gal A precipitated, compared to about 30% (>2×10⁶ U) atpH 5.0. At pH 6.0, the estimated pH of the TGN (Griffith and Simons,1986, Science 234:438-443), about 12% of the enzyme formed particulateaggregates that could be pelleted by centrifugation at 15,000×g. FIG.27B shows that the turbidity, as a measure of aggregation (Halper andStere, 1977, Arch. Biochem. Biophys. 184:529-534), of solutionscontaining 0.1 to 10 mg/ml of α-Gal A at either pH 5.0 or 7.0 increasedas a function of enzyme concentration. Moreover, the turbidity of a 1mg/ml α-Gal A solution was essentially unaffected by the presence ofincreasing albumin concentrations from 0.1 to 10 mg/ml at pH 5.0 (FIG.27B; control). Finally, electrophoresis of the supernatant and pelletfractions from solutions containing α-Gal A (10 mg/ml) and bovine serumalbumin (BSA) (2 mg/ml) incubated at varying hydrogen ion concentrationsrevealed that the increasing precipitation of α-Gal A with decreasing pHwas enzyme specific, as the BSA did not precipitate over this pH range(FIG. 27C).

10.3. DISCUSSION

An "aggregation-secretion" model is proposed to account for thererouting of human α-Gal A as a prototype for overproduced targetedproteins. As depicted in FIG. 28, the overproduced enzyme is normallysynthesized and processed until it reaches the trans-Golgi network(TGN). In this structure, the overproduced enzyme is accumulated andsubjected to a markedly more acidic environment, (app. pH 6.0), whichleads to increased protein-protein interactions that generate solubleand particulate α-Gal A aggregates. As a result of such aggregation, theenzyme's M6P moieties become inaccessible or less accessible for bindingto the MGPR. The aggregates with inaccessible MGP moieties, by default,are rerouted via the constitutive secretory pathway (Helms et al., 1990,J. Biol. Chem. 265:20,027-20,032).

The cellular response, therefore, to the overproduction oflysosome-targeted protein is to transport those proteins havingavailable MGP residues to the lysosome and to reroute the majority ofthe overproduced, and presumably aggregated, proteins through theconstitutive secretion pathway. The fact that large amounts ofrecombinant human α-Gal A are secreted by CHO cells permits thescaled-up production and easy purification of the recombinant enzyme forcrystallography and for trials of enzyme replacement therapy in patientswith Fabry disease. In addition, it is clear that the amplificationseries of overexpressing α-Gal A CHO cells provides a uniqueexperimental mammalian system to efficiently characterize thebiosynthesis, post-translational modifications, and mechanismsresponsible for the lysosomal targeting and selective secretion of thisprototype lysosomal enzyme, thereby providing further insight into thenature of protein transport and sorting in mammalian cells.

Thus, the overexpression of lysosomal and perhaps other targetedproteins in CHO cells provides a convenient method for producing largeamounts of the protein for structural analyses and/or therapeuticapplications, as well as providing a useful approach to study proteinbiosynthesis and sorting.

11. DEPOSIT OF MICROORGANISMS

The following E. coli strains carrying the listed plasmids have beendeposited with the Agricultural Research Culture Collection (NRRL),Peoria, Ill. and have been assigned the following accession number:

    ______________________________________                                        Host Cell                                                                              Strain     Plasmid   Accession No.                                   ______________________________________                                        E. coli  k12        p91.AGA   B 18722                                         E. Coli  k12        pAGA-PA   B 18723                                         ______________________________________                                    

The present invention is not to be limited in scope by themicroorganisms deposited since the deposited embodiments are intended asillustration of individual aspects of the invention and anymicroorganisms, or constructs which are functionally equivalent arewithin the scope of this invention. Indeed various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1393 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..1350                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGGTTAATCTTAAAAGCCCAGGTTACCCGCGGAAATTTATGCTGTCCGGTCACCGTGACA60                ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTT108                           MetGlnLeuA rgAsnProGluLeuHisLeuGlyCysAlaLeuAlaLeu                             151015                                                                        CGCTTCCTGGCCCTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTG156                           ArgPheLeu AlaLeuValSerTrpAspIleProGlyAlaArgAlaLeu                             202530                                                                        GACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAG204                           AspAsnGlyLeu AlaArgThrProThrMetGlyTrpLeuHisTrpGlu                             354045                                                                        CGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATC252                           ArgPheMetCysAsnLe uAspCysGlnGluGluProAspSerCysIle                             505560                                                                        AGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGC300                           SerGluLysLeuPheMetGluMetA laGluLeuMetValSerGluGly                             65707580                                                                      TGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATG348                           TrpLysAspAlaGlyTyrGlu TyrLeuCysIleAspAspCysTrpMet                             859095                                                                        GCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGC396                           AlaProGlnArgAspSerGlu GlyArgLeuGlnAlaAspProGlnArg                             100105110                                                                     TTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGA444                           PheProHisGlyIleArgGlnLe uAlaAsnTyrValHisSerLysGly                             115120125                                                                     CTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGC492                           LeuLysLeuGlyIleTyrAlaAspValG lyAsnLysThrCysAlaGly                             130135140                                                                     TTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCT540                           PheProGlySerPheGlyTyrTyrAspIleAspAla GlnThrPheAla                             145150155160                                                                  GACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGT588                           AspTrpGlyValAspLeuLeuLysPheAspGly CysTyrCysAspSer                             165170175                                                                     TTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAAT636                           LeuGluAsnLeuAlaAspGlyTyrLysHisMe tSerLeuAlaLeuAsn                             180185190                                                                     AGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATG684                           ArgThrGlyArgSerIleValTyrSerCysGluT rpProLeuTyrMet                             195200205                                                                     TGGCCCTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAAT732                           TrpProPheGlnLysProAsnTyrThrGluIleArgGln TyrCysAsn                             210215220                                                                     CACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAG780                           HisTrpArgAsnPheAlaAspIleAspAspSerTrpLysSerIleLys                              225230235240                                                                  AGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTT828                           SerIleLeuAspTrpThrSerPheAsnGlnGluArgIleValAs pVal                             245250255                                                                     GCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAAC876                           AlaGlyProGlyGlyTrpAsnAspProAspMetLeuValIleG lyAsn                             260265270                                                                     TTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCT924                           PheGlyLeuSerTrpAsnGlnGlnValThrGlnMetAlaLeuTrp Ala                             275280285                                                                     ATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGC972                           IleMetAlaAlaProLeuPheMetSerAsnAspLeuArgHisIleSer                               290295300                                                                    CCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCAAT1020                          ProGlnAlaLysAlaLeuLeuGlnAspLysAspValIleAlaIleAsn                              305 310315320                                                                 CAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAAC1068                          GlnAspProLeuGlyLysGlnGlyTyrGlnLeuArgGlnGlyAspAsn                               325330335                                                                    TTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCT1116                          PheGluValTrpGluArgProLeuSerGlyLeuAlaTrpAlaValAla                               340345350                                                                    ATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCA1164                          MetIleAsnArgGlnGluIleGlyGlyProArgSerTyrThrIleAla                              35 5360365                                                                    GTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATC1212                          ValAlaSerLeuGlyLysGlyValAlaCysAsnProAlaCysPheIle                              370 375380                                                                    ACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACT1260                          ThrGlnLeuLeuProValLysArgLysLeuGlyPheTyrGluTrpThr                              385390 395400                                                                 TCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAG1308                          SerArgLeuArgSerHisIleAsnProThrGlyThrValLeuLeuGln                              405 410415                                                                    CTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAAAAAAAAA1357                         LeuGluAsnThrMetGlnMetSerLeuLysAspLeuLeu                                       42042 5430                                                                    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1393                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 429 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlnLeuArgAsnProGluLeuHisLeuGlyCysAlaLeuAlaLeu                              151015                                                                        ArgPheLeuAlaLeuValSerTrpAspIleProGlyAlaArgAlaLeu                               202530                                                                       AspAsnGlyLeuAlaArgThrProThrMetGlyTrpLeuHisTrpGlu                              354045                                                                        ArgPheMetCysAsnLeu AspCysGlnGluGluProAspSerCysIle                             505560                                                                        SerGluLysLeuPheMetGluMetAlaGluLeuMetValSerGluGly                              657075 80                                                                     TrpLysAspAlaGlyTyrGluTyrLeuCysIleAspAspCysTrpMet                              859095                                                                        AlaProGlnArgAspSerGluGlyArgLeuGlnAlaAs pProGlnArg                             100105110                                                                     PheProHisGlyIleArgGlnLeuAlaAsnTyrValHisSerLysGly                              115120125                                                                     LeuLys LeuGlyIleTyrAlaAspValGlyAsnLysThrCysAlaGly                             130135140                                                                     PheProGlySerPheGlyTyrTyrAspIleAspAlaGlnThrPheAla                              145150 155160                                                                 AspTrpGlyValAspLeuLeuLysPheAspGlyCysTyrCysAspSer                              165170175                                                                     LeuGluAsnLeuAlaAspGlyTyrLys HisMetSerLeuAlaLeuAsn                             180185190                                                                     ArgThrGlyArgSerIleValTyrSerCysGluTrpProLeuTyrMet                              195200 205                                                                    TrpProPheGlnLysProAsnTyrThrGluIleArgGlnTyrCysAsn                              210215220                                                                     HisTrpArgAsnPheAlaAspIleAspAspSerTrpLysSerIleLys                              225 230235240                                                                 SerIleLeuAspTrpThrSerPheAsnGlnGluArgIleValAspVal                              245250255                                                                     AlaGlyProGlyGly TrpAsnAspProAspMetLeuValIleGlyAsn                             260265270                                                                     PheGlyLeuSerTrpAsnGlnGlnValThrGlnMetAlaLeuTrpAla                              275280 285                                                                    IleMetAlaAlaProLeuPheMetSerAsnAspLeuArgHisIleSer                              290295300                                                                     ProGlnAlaLysAlaLeuLeuGlnAspLysAspValIleAlaIleAsn                              305310315320                                                                  GlnAspProLeuGlyLysGlnGlyTyrGlnLeuArgGlnGlyAspAsn                              325330335                                                                     PheG luValTrpGluArgProLeuSerGlyLeuAlaTrpAlaValAla                             340345350                                                                     MetIleAsnArgGlnGluIleGlyGlyProArgSerTyrThrIleAla                              355 360365                                                                    ValAlaSerLeuGlyLysGlyValAlaCysAsnProAlaCysPheIle                              370375380                                                                     ThrGlnLeuLeuProValLysArgLysLeuGlyPhe TyrGluTrpThr                             385390395400                                                                  SerArgLeuArgSerHisIleAsnProThrGlyThrValLeuLeuGln                              405410 415                                                                    LeuGluAsnThrMetGlnMetSerLeuLysAspLeuLeu                                       420425                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 411 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                          (ii) MOLECULE TYPE: protein                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetLeuLeuLysThrValLeuLeuLeuGlyHisValAlaGlnValLeu                              151015                                                                        MetLeuAspAsnGlyLeuLeuGlnThrProProMet GlyTrpLeuAla                             202530                                                                        TrpGluArgPheArgCysAsnIleAsnCysAspGluAspProLysAsn                              354045                                                                        CysIle SerGluGlnLeuPheMetGluMetAlaAspArgMetAlaGln                             505560                                                                        AspGlyTrpArgAspMetGlyTyrThrTyrLeuAsnIleAspAspCys                              6570 7580                                                                     TrpIleGlyGlyArgAspAlaSerGlyArgLeuMetProAspProLys                              859095                                                                        ArgPheProHisGlyIleProPheLeuA laAspTyrValHisSerLeu                             100105110                                                                     GlyLeuLysLeuGlyIleTyrAlaAspMetGlyAsnPheThrCysMet                              1151201 25                                                                    GlyTyrProGlyThrThrLeuAspLysValValGlnAspAlaGlnThr                              130135140                                                                     PheAlaGluTrpLysValAspMetLeuLysLeuAspGlyCysPheSer                              145 150155160                                                                 ThrProGluGluArgAlaGlnGlyTyrProLysMetAlaAlaAlaLeu                              165170175                                                                     AsnAlaThrGlyArgPr oIleAlaPheSerCysSerTrpProAlaTyr                             180185190                                                                     GluGlyGlyLeuProProArgValAsnTyrSerLeuLeuAlaAspIle                              195200 205                                                                    CysAsnLeuTrpArgAsnTyrAspAspIleGlnAspSerTrpTrpSer                              210215220                                                                     ValLeuSerIleLeuAsnTrpPheValGluHisGlnAspIleLeuGln                               225230235240                                                                 ProValAlaGlyProGlyHisTrpAsnAspProAspMetLeuLeuIle                              245250255                                                                     GlyAsn PheGlyLeuSerLeuGluGlnArgSerArgAlaGlnMetAla                             260265270                                                                     LeuTrpThrValLeuAlaAlaProLeuLeuMetSerThrAspLeuArg                              275 280285                                                                    ThrIleSerAlaGlnAsnMetAspIleLeuGlnAsnProLeuMetIle                              290295300                                                                     LysIleAsnGlnAspProLeuGlyIleGlnGlyArgIl eHisLysGlu                             305310315320                                                                  LysSerLeuIleGluValTyrMetArgProLeuSerAsnLysAlaSer                              325330 335                                                                    AlaLeuValPhePheSerCysArgThrAspMetProTyrArgTyrHis                              340345350                                                                     SerSerLeuGlyGlnLeuAsnPheThrGlySerValIleTyrGluAla                               355360365                                                                    GlnAspValTyrSerGlyAspIleIleSerGlyLeuArgAspGluThr                              370375380                                                                     AsnPheThrValIleIleAsnProSer GlyValValMetTrpTyrLeu                             385390395400                                                                  TyrProIleLysAsnLeuGluMetSerGlnGln                                             405410                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 404 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetPheAlaPheTyrPheLeuThrAlaCysIleSerLeuLysGlyVal                              15 1015                                                                       PheGlySerTyrAsnGlyLeuGlyLeuThrProGlnMetGlyTrpAsp                              202530                                                                        AsnTrpAsnThrPheAlaCysAspValSerGlu GlnLeuLeuLeuAsp                             354045                                                                        ThrAlaAspArgIleSerAspLeuGlyLeuLysAspMetGlyTyrLys                              505560                                                                        TyrIleIle LeuAspAspCysTrpSerSerGlyArgAspSerAspGly                             65707580                                                                      PheLeuValAlaAspGluGlnLysPheProAsnGlyMetGlyHisVal                               859095                                                                       AlaAspHisLeuHisAsnAsnSerPheLeuPheGlyMetTyrSerSer                              100105110                                                                     AlaGlyGluTyrThrCysAlaGly TyrProGlySerLeuGlyArgGlu                             115120125                                                                     GluGluAspAlaGlnPhePheAlaAsnAsnArgValAspTyrLeuLys                              130135140                                                                     TyrAspAsnCysTyrAsnLysGlyGlnPheGlyThrProGluSerTyr                              145150155160                                                                  ArgLysMetSerAspAlaLeuAsnLysThrGlyArgProIlePheTyr                               165170175                                                                    SerCysAsnTrpGlyLeuTyrGlySerGlyIleAlaAsnSerTrpArg                              180185190                                                                     MetSerGlyAspV alThrAlaGluPheThrArgProAspSerCysPro                             195200205                                                                     AspGlyTyrTyrAlaGlyPheSerIleMetAsnIleLeuAsnLysAla                              210215 220                                                                    AlaProMetGlyGlnAsnAlaGlyValGlyGlyTrpAsnAspLeuAsp                              225230235240                                                                  AsnLeuGluValGlyValGlyAsnLeuThrAspAspGlu GluLysAla                             245250255                                                                     HisPheSerMetTrpAlaMetValLysSerProLeuIleIleGlyAla                              260265270                                                                     As nValAsnAsnLeuLysAlaSerSerTyrSerIleTyrSerGlnAla                             275280285                                                                     SerValIleAlaIleAsnGlnAspSerAsnGlyIleProAlaArgVal                              290 295300                                                                    SerAspThrAspGluTyrGlyGluIleTrpSerGlyProLeuAspAsn                              305310315320                                                                  GlyAspGlnValValAlaLeuLeuAsnG lyGlySerValSerArgPro                             325330335                                                                     MetAsnThrThrLeuGluIleAspSerLeuGlyLysLysLeuThrSer                              340345 350                                                                    ThrAspAspLeuTrpAlaAsnArgValThrAlaSerIleGlyArgLys                              355360365                                                                     ThrGlyLeuTyrGluTyrLysAspGlyLeuLysAsnArgLeuGlyGln                               370375380                                                                    LysGlySerLeuIleLeuAsnValProAlaHisIleAlaPheArgLeu                              385390395400                                                                  ArgProSerSer                                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GluGlnThrIleAlaAspThrLeuGlyProGlyGly                                          15 10                                                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ProSerValIleTyrGlyAsnValArgAsn                                                15 10                                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GluValAlaCysLeuValAspAlaAsnGlyIleGlnPro                                       1 510                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 297 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..279                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      GAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGAACTGTT48                            GluTrpThrSerArgLeuArgSerHisIleAsnProThrGlyThrVal                              1510 15                                                                       TTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTT96                            LeuLeuGlnLeuGluAsnThrMetGlnMetSerLeuLysAspLeuLeu                              2025 30                                                                       CCGGCTGGTCCGGCGCAACACGATGAAGCTCAACAAAATGCTTTTTAT144                           ProAlaGlyProAlaGlnHisAspGluAlaGlnGlnAsnAlaPheTyr                              35404 5                                                                       CAAGTCTTAAATATGCCTAACTTAAATGCTGATCAACGCAATGGTTTT192                           GlnValLeuAsnMetProAsnLeuAsnAlaAspGlnArgAsnGlyPhe                              505560                                                                        ATCCAA AGCCTTAAAGATGATCCAAGCCAAAGTGCTAACGTTTTAGGT240                          IleGlnSerLeuLysAspAspProSerGlnSerAlaAsnValLeuGly                              65707580                                                                      GA AGCTCAAAAACTTAATGACTCTCAAGCTCCAAAATAAGGATCCC286                            GluAlaGlnLysLeuAsnAspSerGlnAlaProLys                                          8590                                                                          GGAATTCGGCC 297                                                               (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GluTrpThrSerArgLeuArgSerHisIleAsnProThrG lyThrVal                             151015                                                                        LeuLeuGlnLeuGluAsnThrMetGlnMetSerLeuLysAspLeuLeu                              202530                                                                        Pro AlaGlyProAlaGlnHisAspGluAlaGlnGlnAsnAlaPheTyr                             354045                                                                        GlnValLeuAsnMetProAsnLeuAsnAlaAspGlnArgAsnGlyPhe                              50 5560                                                                       IleGlnSerLeuLysAspAspProSerGlnSerAlaAsnValLeuGly                              65707580                                                                      GluAlaGlnLysLeuAsnAspSerGlnAl aProLys                                         8590                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCGAATTCATGCTGTCCGG TCACCGTG27                                                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGCCGGACCAGCCGGAA GTAAGTCTTTTAATG32                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCGGCTGGTCCGGCG CAACACGATGAAGCT30                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGCCGAATTCCGG GATCCTTATTTTGGAGCTTGAGA36                                   

What is claimed is:
 1. A method for producing human galactosidase Acomprising:(a) culturing a transformed mammalian cell with β-galactosideα2,6-sialyltransferase gene which expresses β-galactosideα2,6-sialyltransferase such that the cell is capable of peptidesialylation, further containing a heterologous chromosomally integratednucleotide sequence encoding human α-galactosidase A operativelyassociated with a nucleotide sequence that regulates gene expression anda selectable marker controlled by the same or a different regulatorysequence, so that the α-galactosidase A nucleotide sequence is stablyoverexpressed and an enzymatically active, sialylated glycoform of theα-galactosidase A enzyme is secreted by the mammalian cell; and (b)isolating enzymatically active α-galactosidase A enzyme from themammalian cell culture.
 2. The method according to claim 1 in which thenucleotide sequence encoding α-galactosidase A comprises the sequencedepicted in FIG. 1A from nucleotide number 1 to 1299 [SEQ ID NO:1]. 3.The method according to claim 1 in which the nucleotide sequenceencoding α-galactosidase A comprises the sequence depicted in FIG. 1Afrom nucleotide number 91 to 1299 [SEQ ID NO:1].
 4. The method accordingto claim 1 in which the nucleotide sequence that regulates geneexpression comprises a viral promoter.
 5. The method according to claim1 in which the nucleotide sequence that regulates gene expressioncomprises an inducible promoter.
 6. The method according to claim 1 inwhich the mammalian cell is a Chinese hamster ovary cell line.
 7. Themethod according to claim 1 wherein, in the presence of selection, thechromosomally integrated nucleotide sequences are amplified.
 8. Themethod according to claim 1 in which the selectable marker isdihydrofolate reductase.
 9. The method according to claim 7 in which theselectable marker is dihydrofolate reductase and the selection ismethotrexate.